Transcript
User Bulletin ABI 373 DNA Sequencer (for Rhodamine Terminator Users), ABI 373 DNA Sequencer with BigDye Filter Wheel Upgrade (for BigDye Terminator and dRhodamine Terminator Users), ABI PRISM 377 DNA Sequencer, and ABI PRISM 310 Genetic Analyzer March 30, 1998
SUBJECT: Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Introduction
The precipitation methods presented in this user bulletin are listed below. All of the protocols work well when they are performed exactly as written, using the recommended type and concentration of reagents. Several protocols for each sequencing chemistry are presented to offer a choice in terms of reagents and process. Ideally, we suggest performing controlled reactions with each method to determine the one that works best for you. If this is not possible, start with the first protocol listed below for the type of chemistry you are using. If residual dyes persist, try an alternative protocol.
Chemistry BigDye™ Terminators
Using Recommended Using Microcentrifuge Precipitation Methods MicroAmp Trays Tubes 60% +/– 5% Isopropanol
page 19
page 21
60% +/– 3% Ethanol
page 23
page 25
Ethanol/Sodium Acetate
page 27
page 29
dRhodamine Ethanol/Magnesium Chloride and Rhodamine Ethanol/Sodium Acetate Terminators Shrimp Alkaline Phosphatase
page 31
page 33
page 35
page 37
page 39
page 41
Table of Contents
The following topics are discussed in this user bulletin: Topic Sequencing Kits and Instruments
See page 3
Factors to Consider With Precipitation Methods
4
Location of Residual Dye Terminators
6
Technical Tips
11
Importance of Isopropanol Concentration for BigDye Terminator Precipitations
13
Importance of the Ethanol Concentration for BigDye Terminator Precipitations
15
Ethanol Precipitation with MgCl2 Not Recommended for BigDye Terminators
16
Importance of Washing Samples in Microcentrifuge Tubes
17
Effect of Spin Speeds and Times on Precipitations
18
ABI PRISM™ BigDye™ Terminator Precipitation Protocols ♦
60% +/–5% Isopropanol Method in MicroAmp® Trays
19
♦
60% +/– 5% Isopropanol Method in Microcentrifuge Tubes
21
♦
60% +/– 3% Ethanol Method in MicroAmp Trays
23
♦
60% +/– 3% Ethanol Method in Microcentrifuge Tubes
25
♦
Ethanol/Sodium Acetate (NaOAc) Method in MicroAmp Trays
27
♦
Ethanol/Sodium Acetate (NaOAc) Method in Microcentrifuge Tubes
29
ABI PRISM™ dRhodamine and ABI PRISM™ Rhodamine Dye Terminator Precipitation Protocols ♦
Ethanol/ Magnesium Chloride (MgCl2) Method in MicroAmp Trays
31
♦
Ethanol/ Magnesium Chloride (MgCl2) Method in Microcentrifuge Tubes
33
♦
Ethanol/Sodium Acetate (NaOAc) Method in MicroAmp Trays
35
♦
Ethanol/Sodium Acetate (NaOAc) Method in Microcentrifuge Tubes
37
♦
Shrimp Alkaline Phosphatase (SAP) Digestion Method in MicroAmp Trays
39
♦
SAP Method in Microcentrifuge Tubes
41
Page 2 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Sequencing Kits and Instruments Relevant Sequencing Kits
The following PE Applied Biosystems sequencing kits are relevant to this document: Chemistry
Part Numbers
ABI PRISM™ BigDye™ Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS
4303149 for the 100 reaction kit 4303150 for the 1000 reaction kit
ABI PRISM™ dRhodamine Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS
403044 for the 100 reaction kit 403045 for the 1000 reaction kit
ABI PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA Polymerase, FS
Compatibility of Instruments and Sequencing Chemistries
402080 for the 100 reaction kit
The following table indicates the compatibility of each terminator chemistry with ABI PRISM ® 377, 310, and ABI™ 373 Instruments. Instrument
Terminator Chemistry
ABI PRISM ABI PRISM 377 310
ABI 373 with BDFW*
ABI 373
BigDye Terminator
✓
✓
✓
—
dRhodamine Terminator
✓
✓
✓
—
Rhodamine Terminator
✓
✓
—
✓
* BDFW = BigDye Filter Wheel ✓ = Compatible with this instrument — = Not compatible with this instrument
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 3 of 42
Factors to Consider With Precipitation Methods Important Considerations
Variable-Speed Centrifuge and MicroAmp Trays
Several factors must be considered when using any of the precipitation methods described in this user bulletin. The most important factors are: ♦
Careful adherence to protocol instructions
♦
Concentration variability of ethanol or isopropanol
♦
Type of tray, tube, and centrifuge used
♦
Centrifuge time and g force
The protocols designed for use with 96-well MicroAmp trays require the samples be centrifuged at different speeds: the first spin at > 2000 x g for 30 minutes to retrieve sequencing extension products, and the inverted spin at 700 x g for 1 minute to remove any remaining ethanol after decanting. Therefore, a variable-speed centrifuge functioning at the recommended spin speeds is essential for the success of these protocols. Calculating Centrifuge Speed Use this formula to calculate centrifuge spin speeds:
g = 11.18 x r x (rpm/1000)2 where:
g = relative centrifugal force rpm = revolutions per minute r = radius of the rotor in cm
Type of Ethanol or Isopropanol Used
Ethanol To eliminate variability, we recommend using commercially prepared 95% non-denatured ethanol, American Chemical Society (ACS) reagent grade. Although it is relatively easy to mix bulk 95% non-denatured ethanol, be careful when reconstituting from absolute ethanol. Absolute ethanol takes in atmospheric moisture and, over time, will no longer be 100% ethanol. Even slight differences in concentration can affect results.
Page 4 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Isopropanol Use only isopropanol (2-propanol, anhydrous). Do not use n-propanol.
96-well MicroAmp Trays versus Microcentrifuge Tubes
Instructions for each precipitation method listed in this user bulletin are presented twice. The first protocol listed for a particular method is designed for use with PE Applied Biosystems MicroAmp ® 96-well trays. The second protocol listed for the same method is designed for use with microcentrifuge tubes. 96-well Trays The protocols designed for use with 96-well trays were optimized using PE Applied Biosystems MicroAmp trays. Other 96-well formats should work efficiently, though the maximum amount of ethanol or isopropanol that can be added to each tube may be limited. Some 96-well trays accommodate larger volumes than others. The trays used must tolerate spin speeds up to 3000 x g for at least 30 minutes. Microcentrifuge Tubes Use 1.5 or 0.5 mL microcentrifuge tubes. The following steps are critical when using these tubes: ♦
For ethanol precipitations: rinse the pellet with 70% ethanol
♦
For isopropanol precipitations: rinse the pellet with 75% isopropanol
♦
Removal of all of the ethanol or isopropanol after the first spin using a pipettor rather than decanting
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 5 of 42
Location of Residual Dye Terminators Overview
The protocols presented in this user bulletin are robust and your results will be consistent if the: ♦
Protocols are performed exactly as written
♦
Reagents used are of the type and concentration specified
The following examples show where residual dyes are located when the protocols are not followed exactly, and when the reagents used deviate from what is specified.
Rhodamine Terminator Residual Dyes
Figure 1 on page 7 is a gel image from an ABI PRISM ® 377 DNA Sequencer that shows the results of sub-optimal ethanol precipitation. Because the final ethanol concentration was too high (> 60%), excess rhodamine dyes were precipitated. Residual ethanol or isopropanol in the tubes remaining after aspiration also results in excess dyes. Residual rhodamine dyes generally appear at the beginning of the sequence, and affect data quality up to 50 bases. Figure 2 on page 7 shows how the sequencing analysis software analyzes the regions in Figure 1 with excess rhodamine dye.
Page 6 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
4
3
2
1
Figure 1. Excess rhodamine dye from sub-optimal ethanol precipitation
1
2
3
4
Figure 2. Excess rhodamine dye as analyzed by sequencing analysis software. The numbered peaks on this electropherogram correspond to the numbered residual dye peaks in Figure 1.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 7 of 42
dRhodamine Terminator Residual Dyes
Figure 3 is a gel image from an ABI PRISM 377 DNA Sequencer that shows the results of sub-optimal ethanol precipitation. Because the final ethanol concentration was too high (> 60%), excess dRhodamine dyes were precipitated. Residual ethanol or isopropanol in the tubes remaining after aspiration also results in excess dyes. Residual dRhodamine dyes generally appear at the beginning of the sequence, and affect data quality up to 50 bases. Figure 4 on page 9 shows how the sequencing analysis software analyzes the regions in Figure 3 with excess dRhodamine dye.
4
3 2
1
Figure 3. Excess dRhodamine dye from sub-optimal ethanol precipitation
Page 8 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
1
23
4
Figure 4. Excess dRhodamine dye as analyzed by sequencing analysis software. The numbered peaks on this electropherogram correspond to the numbered residual dyes in Figure 3 on page 8.
BigDye Terminator Residual Dyes
The gel image shown in Figure 5 on page 10 is from an ABI PRISM 377 DNA Sequencer, and shows the results of sub-optimal ethanol precipitation. Because the final ethanol concentration was too high (> 63%), excess BigDye terminators were precipitated. Residual ethanol or isopropanol in the tubes remaining after aspiration also results in excess dyes. If not removed by precipitation, residual dye in location 1 will not affect base calling accuracy, but can result in a false start point during raw data analysis. Residual BigDye terminators in locations 2–7 generally affect the base calling accuracy of the first 40 bases, while residual dye in location 8 usually appears between 90 and 100 bases. The electropherogram in Figure 6 on page 10 shows the location of excess dyes in analyzed data. If the precipitation protocols are followed properly, residual dye locations 1, 5, 6, 7, and 8 are removed completely. Occasionally, residual dye in the areas marked 2, 3, and 4 show up even when the protocols are performed carefully. The energy transfer component of BigDye terminators means that a very small amount of residual terminator can be detected. Only the first 10 bases are affected. If your application requires obtaining sequence immediately after the primer, it may be more appropriate to use CentriSep spin columns.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 9 of 42
7
6 5 4 3 2 8
1
Figure 5. Excess BigDye terminators from sub-optimal ethanol precipitation
1
2
3
4 5
6
7
8
Figure 6. Excess BigDye terminators as analyzed by sequencing analysis software. The numbered peaks on this electropherogram correspond to the numbered residual dye locations in Figure 5.
Page 10 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Technical Tips Tips for Optimal Protocol Performance
Following this list of suggestions will help ensure optimal protocol performance. ♦
If reactions are scaled down to 10 µL or less in MicroAmp tubes, we recommend still performing the precipitation in a 100 µL final volume instead of 50 µL. See the following example.
Half Volume Reaction (in µL)
Standard Volume Reaction (in µL)
Dye terminator reaction
10 µL
20
Add 100% isopropanol
60 µL
60
Add deionized water
30 µL
20
Total volume
100 µL
100
Precipitation Component
Larger volumes dilute out excess dye terminators, making their removal more efficient. ♦
After the addition of ethanol or isopropanol, ensure that the solvent thoroughly mixes with the aqueous sample.
♦
After the addition of ethanol or isopropanol, let the samples sit at room temperature for at least 15 minutes for maximum precipitation of extension fragments.
♦
When using MicroAmp trays, invert spin immediately after the 30 minute spin. When using microcentrifuge tubes, aspirate off the supernatant immediately after the 20 minute spin. If the inverted spin or aspiration is not performed immediately, the pellets may loosen up and be lost during the spin or aspiration, resulting in weak signals. If the pellet is left in ethanol or isopropanol for more than one minute, spin the samples again at:
♦
–
A spin speed greater than 2000 x g for 5 minutes for MicroAmp trays.
–
Maximum speed for 5 minutes for microcentrifuge tubes.
When using microcentrifuge tubes, aspirating off the supernatant is more effective than decanting. If performed carefully, aspiration removes practically all of the supernatant.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 11 of 42
♦
If your centrifuge reaches only about 1400 x g, spin the samples for at least 45 minutes. We cannot guarantee that all extension products are recoverable at spin speeds of 1400 x g.
♦
Work from a fresh source of 95% ethanol. Repeated opening can lead to ethanol evaporation and an ethanol concentration lower than 95%. Extension fragments are then difficult to precipitate, resulting in weak signals.
♦
For methods using ethanol, use non-denatured ethanol. Denatured ethanol may contain additives such as benzene, toluene, methanol, etc. that may fluoresce during sequencing.
♦
A sequencing reaction that has produced weak signals may show more residual dye. This is because the sequencing analysis software scales up the weak peak signals, resulting in increased residual dye peaks.
Page 12 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Importance of Isopropanol Concentration for BigDye Terminator Precipitations Overview
BigDye terminators produce greater signal intensity than rhodamine and dRhodamine dye terminators. Therefore, any residual BigDye terminators will be readily visible. Figure 7 on page 14 is a gel image from an ABI PRISM 377. MicroAmp trays and a variable-speed centrifuge were used. Figure 7 shows how deviations from the recommended final isopropanol concentration of 60% +/– 5% will affect precipitation results. A 70% final isopropanol concentration (lane A) resulted in strong signals but excessive dye. A 50% final isopropanol concentration (lane E) resulted in less residual dye but no precipitation of extension fragments. Generally, a final isopropanol concentration of 60% +/– 5% removes most excess BigDye terminators without loss of signals. The first three residual dye areas shown in lanes B, C, and D affect the first 10 bases. If these first 10 bases are critical for your application (for example, primer walking), we recommend using CentriSep spin columns to remove all excess dyes. Another option is precipitating in larger volumes, and rinsing with larger volumes of 75% isopropanol. A larger volume precipitation results in greater dilution of excess dye terminators. Since the residual supernatant after the first spin contains the excess dye, it will be less problematic if it is more dilute. If a wash step is also performed, residual dye problems should be minimal or absent.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 13 of 42
A
B
C
D
E A = 70% Isopropanol B = 65% Isopropanol C = 60% Isopropanol D = 55% Isopropanol E = 50% Isopropanol
Figure 7. Gel image representing the importance of the final concentration of isopropanol in the precipitation of BigDye Terminators using MicroAmp trays. Recommended final isopropanol concentration is 60% +/– 5% (lanes B, C, and D).
Page 14 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Importance of the Ethanol Concentration for BigDye Terminator Precipitations Overview
The optimum final concentration of ethanol for BigDye Terminator precipitations is 60% +/– 3%. Slight differences in the final concentration result in increased residual dyes. When exposed to air, absolute ethanol pulls moisture and becomes more dilute. This causes slight variations in concentration when 95% ethanol is reconstituted. Therefore, we recommend using commercially prepared 95% ethanol to eliminate variations. As shown in lane B, a significant amount of residual dye is still present when the final ethanol concentration is 65%. A final ethanol concentration of 63% (data not shown) removes most residual dye without affecting signal. At a 55% final concentration (lane D), extension fragments become difficult to precipitate. Increasing the final concentration to 57% (data not shown) removes residual dye while precipitating extension fragments efficiently. A
B
C
D A = 70% EtOH B = 65% EtOH C = 60% EtOH D = 55% EtOH
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 15 of 42
Ethanol Precipitation with MgCl2 Not Recommended for BigDye Terminators Overview
The gel image below shows what can happen when the wrong protocol is used. Ethanol precipitation with MgCl2 is not recommended for BigDye Terminators because it precipitates excess BigDyes (lane A). Removing nearly all residual dyes when MgCl2 is added (lane B) requires an extra 70% wash. As shown in lane C, salt is not necessary to precipitate BigDye Terminator extension fragments. The same signal strengths are obtained with or without the addition of salt. This precipitation experiment was done using MicroAmp trays. A
B
C
D A = 60% EtOH + 0.5mM MgCl2 B = 60% EtOH + 0.5mM MgCl2 with 70% wash C = 60% EtOH D = CentriSep spin column
Page 16 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Importance of Washing Samples in Microcentrifuge Tubes Overview
When using microcentrifuge tubes, isopropanol precipitations require a 75% isopropanol wash, and ethanol precipitations require a 70% ethanol wash. The residual dyes shown in lanes A and B below result from isopropanol precipitation without a rinse after the first aspiration. Excess dyes are soluble in isopropanol; therefore, residual solvent contributes to residual dyes. The 75% wash removes residual isopropanol from the tube walls and surface of the pellet. Lanes C and D result from rinsing with 75% isopropanol immediately after the first aspiration. The same results apply to ethanol precipitations as shown in lanes E, F, G, and H. Aspirating off the supernatant is more effective than decanting when using microcentrifuge tubes. If performed carefully, aspiration removes nearly all of the supernatant. A
B
C
D
E
F
G
H A = 60% isopropanol B = 60% isopropanol C = 60% isopropanol + 75% wash D = 60% isopropanol + 75% wash E = 60% EtOH F = 60% EtOH G = 60% EtOH + 70% wash H = 60% EtOH + 70% wash
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 17 of 42
Effect of Spin Speeds and Times on Precipitations Overview
Spin speeds and times are key factors in the success of ethanol and isopropanol precipitations. Lanes A and B in the figure below show precipitations in MicroAmp trays spun at 3000 x g for 20 minutes. Lanes C and D show precipitations in MicroAmp trays spun at 3000 x g for 30 minutes. Signal loss occurred when the spin time was reduced to 20 minutes, rather than 30 minutes as is directed in the protocols for MicroAmp trays. We recommend: ♦
30 minutes at spin speeds >2000 x g for MicroAmp trays
♦
45 minutes at spin speeds of 1400—2000 x g for MicroAmp trays
♦
20 minutes at maximum speed for microcentrifuge tubes
A formula for calculating spin speeds is listed under “Variable-Speed Centrifuge and MicroAmp Trays” on page 4. A
B
C
D A = 20 minutes B = 20 minutes C = 30 minutes D = 30 minutes
Page 18 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
ABI PRISM™ BigDye™ Terminator Precipitation Protocols Introduction
60% +/–5% Isopropanol Method in MicroAmp ® Trays (BigDye)
We recommend the following protocols for precipitations in MicroAmp 96-well trays, and 1.5 or 0.5 mL microcentrifuge tubes. Reagents and Equipment Required No salt is required for this precipitation method. ♦
100% isopropanol (2-propanol, anhydrous) at room temperature
IMPORTANT
Use only 2-propanol (anhydrous). Do not use n-propanol.
♦
MicroAmp trays (96-well) and tubes
♦
Pipettor with tips, Gilson Pipetman P200 (Rainin Instruments)
♦
Strip caps or adhesive aluminum tape (3M Scotch Tape 425-3)
IMPORTANT Other types of aluminum tape have not been tested. Tubes must be sealed completely with strip caps or aluminum tape.
♦
Variable speed centrifuge with microtiter plate tray holders capable of reaching spin speed of at least 1400 x g (see “Calculating Centrifuge Speed” on page 4)
Isopropanol Method in MicroAmp Trays (BigDye): Step
Action
1
Remove the MicroAmp tray from the thermal cycler, and remove the caps from each tube.
2
Add the following reagents to the 20 µL reaction for a final isopropanol concentration of 60% +/– 5%: ♦
20 µL of deionized water
♦
60 µL of 100% isopropanol (2-propanol, anhydrous, room temperature)
! WARNING ! CHEMICAL HAZARD. Isopropyl alcohol can be harmful if inhaled, ingested, or absorbed through the skin. It can cause CNS depression, and be irritating to the eyes, skin, and mucous membranes. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3
Seal the tubes with strip caps or aluminum tape (3M Scotch Tape 425-3), and mix by vortexing or inverting the tray several times.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 19 of 42
Isopropanol Method in MicroAmp Trays (BigDye): (continued) Step 4
Action Allow the capped tubes to sit at room temperature for 15 minutes. Note Precipitation times <15 minutes result in loss of short extension products; times >24 hours result in increased precipitation of unincorporated dyes.
5
Place the capped tubes in a tray and the tray in the centrifuge.
6
Centrifuge the tray for 30 minutes at the maximum speed (at least 2000 x g, but not greater than 3000 x g). IMPORTANT For spin speeds between 1400—2000 x g, increase the spin time to 45 minutes to ensure good precipitation. Note MicroAmp tubes in MicroAmp trays can withstand 3000 x g for 30 minutes.
7
Remove the tray, and remove the tape or caps from the tubes without disturbing the pellets.
8
Immediately invert the tray on a paper towel, and place it in the centrifuge again.
9
Spin inverted at 700 x g for 1 minute. IMPORTANT The inverted spin must be performed immediately after centrifugation or loss of signal results. If the tray sits for more than 1 minute before the inverted spin, spin it for an additional 5 minutes at 2000 x g to guarantee recovery of the sample. Removing all residual ethanol during the inverted spin is essential. Note
10
It is not necessary to dry the pellet before resuspension.
Resuspend samples using the appropriate loading solutions for your instrument.
Page 20 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
60% +/– 5% Isopropanol Method in Microcentrifuge Tubes (BigDye)
Reagents and Equipment Required No salt is required for this precipitation method. ♦
Microcentrifuge tubes, 1.5 or 0.5 mL
♦
Microcentrifuge
♦
Vacuum centrifuge or heat block at 90°C
♦
75% isopropanol (2-propanol) at room temperature
♦
100% isopropanol (2-propanol, anhydrous) at room temperature
♦
Pipettor with tips, Gilson Pipetman P200 (Rainin Instruments)
IMPORTANT
Use only 2-propanol (anhydrous). Do not use n-propanol.
Note If precipitations are performed on reduced volume sequencing reactions (10 µL), bring the final volume to 20 µL with deionized water, and then follow the protocol below. If residual dyes are still present, increase the precipitation volume by increasing each component proportionally.
Isopropanol Method in Microcentrifuge Tubes (BigDye): Step
Action
1
After completion of the sequencing reaction, transfer the extension products (20 µL) to microcentrifuge tubes (0.5 or 1.5 mL).
2
Add one of the following reagents to the tubes: ♦
80 µL of 75% isopropanol (2-propanol) or
♦
20 µL of deionized water and 60 µL of 100% isopropanol (2-propanol, anhydrous)
! WARNING ! CHEMICAL HAZARD. Isopropyl alcohol can be harmful if inhaled, ingested, or absorbed through the skin. It can cause CNS depression, and be irritating to the eyes, skin, and mucous membranes. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3
Cap the tubes, and vortex briefly.
4
Leave the tubes at room temperature for 15 minutes to precipitate the extension products.
5
Place the capped tubes in a microcentrifuge, and mark the orientation of the tubes.
6
Spin the tubes for 20 minutes at maximum speed. IMPORTANT
Perform step 7 immediately after this step.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 21 of 42
Isopropanol Method in Microcentrifuge Tubes (BigDye): (continued) Step 7
Action Without disturbing the pellet, carefully aspirate off the supernatant with a pipettor and discard it. IMPORTANT The pellet may or may not be visible. The supernatant must be completely removed because unincorporated dye-labeled terminators are dissolved in the supernatant. The more residual supernatant left in the tube, the more residual terminators are in the sample when it is dried. This results in larger terminator peaks during electrophoresis. Using a pipettor to remove the supernatant is better than decanting.
8
Rinse the pellet with 250 µL of 75% isopropanol (2-propanol), and cap the tubes.
9
Vortex briefly.
10
Centrifuge for 5 minutes at maximum speed.
11
Without disturbing the pellet, carefully aspirate the supernatant with a pipettor and discard.
12
Dry the pellet in a vacuum centrifuge for 10–15 minutes, or remove the caps and place the tubes in a heat block at 90°C for 1 minute. Note Do not over-dry, or the samples may be difficult to resuspend.
13
Resuspend samples using the appropriate loading solutions for your instrument.
Page 22 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
60% +/– 3% Ethanol Method in MicroAmp Trays (BigDye)
Reagents and Equipment Required No salt is required for this precipitation method. ♦
95% non-denatured ethanol (EtOH), American Chemical Society (ACS) reagent grade at room temperature
IMPORTANT 95% EtOH made from absolute EtOH can be inaccurate if the absolute EtOH bottle has pulled atmospheric water. We recommend purchasing commercially prepared 95% EtOH to eliminate variability.
♦
MicroAmp trays (96-well) and tubes
♦
Pipettor with tips, Gilson Pipetman P200 (Rainin Instruments)
♦
Strip caps or adhesive aluminum tape (3M Scotch Tape 425-3)
IMPORTANT Other types of aluminum tape have not been tested. Tubes must be sealed completely with strip caps or aluminum tape.
♦
Variable speed centrifuge with microtiter plate tray holders capable of reaching spin speed of at least 1400 x g (see “Calculating Centrifuge Speed” on page 4)
EtOH Method in MicroAmp Trays (BigDye): Step
Action
1
Remove the MicroAmp tray from the thermal cycler, and remove the cap from each tube.
2
Add the following reagents to the 20 µL reaction for a final EtOH concentration of 60% +/– 3%: ♦
16 µL of deionized water
♦
64 µL of non-denatured 95% ethanol (EtOH; ACS reagent grade) at room temperature
! WARNING ! CHEMICAL HAZARD. Ethanol is a flammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3
Seal the tubes with strip caps or aluminum tape (3M Scotch Tape 425-3), and mix by vortexing or inverting the tray several times.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 23 of 42
EtOH Method in MicroAmp Trays (BigDye): Step 4
Action Allow the capped tubes to sit at room temperature for 15 minutes. Note Precipitation times <15 minutes result in loss of short extension products; times >24 hours result in increased precipitation of unincorporated dyes.
5
Place the capped tubes in a tray and the tray in a centrifuge.
6
Centrifuge the tray for 30 minutes at the maximum speed (at least 2000 x g, but not greater than 3000 x g). IMPORTANT For spin speeds between 1400—2000 x g, increase the spin time to 45 minutes to ensure good precipitation. Note MicroAmp tubes in MicroAmp trays can withstand 3000 x g for 30 minutes.
7
Remove the tray, and remove the tape or caps from the tubes without disturbing the pellets.
8
Immediately invert the tray on a paper towel, and place it in the centrifuge again.
9
Spin inverted at 700 x g for 1 minute. IMPORTANT The inverted spin must be performed immediately after centrifugation or loss of signal results. If the tray sits for more than 1 minute before the inverted spin, spin it for an additional 5 minutes at 2000 x g to guarantee recovery of the sample. Removing all residual ethanol during the inverted spin is essential. Note
10
It is not necessary to dry the pellet before resuspension.
Resuspend samples using the appropriate loading solutions for your instrument.
Page 24 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
60% +/– 3% Ethanol Method in Microcentrifuge Tubes (BigDye)
Reagents and Equipment Required No salt is required for this precipitation method. ♦
Microcentrifuge tubes, 1.5 or 0.5 mL
♦
Microcentrifuge
♦
Vacuum centrifuge or heat block at 90°C
♦
Pipettor with tips, Gilson Pipetman P200 (Rainin Instruments)
♦
70% EtOH (ACS reagent grade) at room temperature
♦
95% EtOH (ACS reagent grade) at room temperature
IMPORTANT 95% EtOH made from absolute EtOH can be inaccurate if the absolute EtOH bottle has pulled atmospheric water. We recommend purchasing commercially prepared 95% EtOH to eliminate variability. Note If precipitations are performed on reduced volume sequencing reactions (10 µL), bring the final volume to 20 µL with deionized water, and then follow the protocol below. If residual dyes are still present, increase the precipitation volume by increasing each component proportionally.
EtOH Method in Microcentrifuge Tubes (BigDye): Step
Action
1
After completion of the sequencing reaction, transfer the extension products (20 µL) to 1.5 or 0.5 mL microcentrifuge tubes.
2
Add the following reagents to the 20 µL reaction: ♦
16 µL deionized water
♦
64 µL non-denatured 95% ethanol at room temperature
! WARNING ! CHEMICAL HAZARD. Ethanol is a flammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3
Cap the tubes, and vortex briefly.
4
Leave the tubes at room temperature for 15 minutes to precipitate the extension products.
5
Place the capped tubes in a microcentrifuge, and mark the orientation of the tubes.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 25 of 42
EtOH Method in Microcentrifuge Tubes (BigDye): Step 6
Action Spin the tubes for 20 minutes at maximum speed. IMPORTANT
7
Perform step 7 immediately after this step.
Without disturbing the pellet, carefully aspirate the supernatant with a pipettor and discard it. IMPORTANT The pellet may or may not be visible. The supernatant must be completely removed because unincorporated dye-labeled terminators are dissolved in the supernatant. The more residual supernatant left in the tube, the more residual terminators are in the sample when it is dried. This results in larger terminator peaks during electrophoresis. Using a pipettor to remove the supernatant is better than decanting.
8
Rinse the pellet with 250 µL of 70% EtOH, and cap the tubes.
9
Vortex briefly.
10
Centrifuge for 5 minutes at the maximum speed.
11
Without disturbing the pellet, carefully aspirate the supernatant with a pipettor and discard.
12
Dry the pellet in a vacuum centrifuge for 10–15 minutes, or remove the caps and place the tubes in a heat block at 90 ˚C for 1 minute. Note Do not over-dry or the samples may be difficult to resuspend.
13
Resuspend samples using the appropriate loading solutions for your instrument.
Page 26 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Ethanol/Sodium Acetate (NaOAc) Method in MicroAmp Trays (BigDye)
Reagents and Equipment Required ♦ 95% non-denatured EtOH (ACS reagent grade) at room temperature IMPORTANT 95% EtOH made from absolute EtOH can be inaccurate if the absolute EtOH bottle has pulled atmospheric water. We recommend purchasing commercially prepared 95% ethanol to eliminate variability.
♦
70% EtOH at room temperature
♦
3M sodium acetate (NaOAc), pH 4.6 (P/N 400320)
♦
MicroAmp trays (96-well) and tubes
♦
Pipettor with tips, Gilson Pipetman P200 (Rainin Instruments)
♦
Strip caps or adhesive aluminum tape (3M Scotch Tape 425-3)
IMPORTANT Other types of aluminum tape have not been tested. Tubes must be sealed completely with strip caps or aluminum tape.
♦
Variable speed centrifuge with microtiter plate tray holders capable of reaching spin speed of at least 1400 x g (see “Calculating Centrifuge Speed” on page 4)
EtOH/NaOAc Method in MicroAmp Trays (BigDye): Step
Action
1
Remove the MicroAmp tray from the thermal cycler, and remove the cap from each tube.
2
Add the following reagents to the 20 µL reaction: ♦
2.0 µL of 3M NaOAc
♦
50 µL of 95% EtOH at room temperature
! WARNING ! CHEMICAL HAZARD. Ethanol is a flammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3
Seal the tubes with strip caps or aluminum tape (3M Scotch Tape 425-3), and mix by vortexing or inverting the tray several times.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 27 of 42
EtOH/NaOAc Method in MicroAmp Trays (BigDye): Step 4
Action Allow the capped tubes to sit at room temperature for 15 minutes. Note Precipitation times <15 minutes result in loss of short extension products; times >24 hours results in increased precipitation of unincorporated dyes.
5
Place the capped tubes in a tray and the tray in a centrifuge.
6
Centrifuge the tray for 30 minutes at the maximum speed (at least 2000 x g, but not greater than 3000 x g). IMPORTANT For spin speeds between 1400—2000 x g, increase the spin time to 45 minutes to ensure good precipitation. Note MicroAmp tubes in MicroAmp trays can withstand 3000 x g for 30 minutes.
7
Remove the tray from the centrifuge and the tape or caps from the tubes without disturbing the pellets.
8
Immediately invert the tray on a paper towel, and tap lightly to decant the ethanol.
9
Add 150 µL of 70% EtOH to rinse the pellets and remove the salt.
10
Cap or seal the tubes, and invert the tray a few times to mix.
11
Spin the tray for 10 minutes at maximum speed (at least 1400 x g but not greater than 3000 x g).
12
Spin inverted at 700 x g for 1 minute. IMPORTANT The inverted spin must be performed immediately after centrifugation or loss of signal results. If the tray sits for more than 1 minute before the inverted spin, spin it for an additional 5 minutes at 2000 x g to guarantee recovery of the sample. Removing all residual ethanol during the invert spin is essential. Note
13
It is not necessary to dry the pellet before resuspension
Resuspend samples using the appropriate loading solutions for your instrument.
Page 28 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Ethanol/Sodium Acetate (NaOAc) Method in Microcentrifuge Tubes (BigDye)
Reagents and Equipment Required ♦ Microcentrifuge tubes, 1.5 or 0.5 mL ♦
Microcentrifuge
♦
Pipettor with tips, Gilson Pipetman P200 (Rainin Instruments)
♦
Vacuum centrifuge or heat block at 90°C
♦
70% EtOH at room temperature
♦
3M NaOAc, pH 4.6 (P/N 400320)
♦
95% non-denatured EtOH (ACS reagent grade) at room temperature
IMPORTANT 95% EtOH made from absolute EtOH can be inaccurate if the absolute EtOH bottle has pulled atmospheric water. We recommend purchasing commercially prepared 95% EtOH to eliminate variability. Note If precipitations are performed on reduced volume sequencing reactions (10 µL), bring the final volume to 20 µL with deionized water, and then follow the protocol below. If residual dyes are still present, increase the precipitation volume by increasing each component proportionally.
EtOH/NaOAc Method in Microcentrifuge Tubes (BigDye): Step
Action
1
After completion of the sequencing reaction, transfer the extension products (20 µL) to 1.5 or 0.5 mL microcentrifuge tubes.
2
Add the following reagents to the tubes: ♦
2 µL of 3M NaOAc, pH 4.6
♦
50 µL of 95% EtOH at room temperature
! WARNING ! CHEMICAL HAZARD. Ethanol is a flammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3
Cap the tubes, and vortex briefly.
4
Leave the tubes at room temperature for 15 minutes to precipitate the extension products.
5
Place the capped tubes in a microcentrifuge, and mark the orientation of the tubes.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 29 of 42
EtOH/NaOAc Method in Microcentrifuge Tubes (BigDye): Step 6
Action Spin the tubes for 20 minutes at maximum speed. IMPORTANT
7
Perform step 7 immediately after this step.
Without disturbing the pellet, carefully aspirate the supernatant with a pipettor and discard it. IMPORTANT The pellet may or may not be visible. The supernatant must be completely removed because unincorporated dye-labeled terminators are dissolved in the supernatant. The more residual supernatant in the tube, the more residual terminators are in the sample when it is dried. This results in larger terminator peaks in the sample during electrophoresis. Using a pipettor to remove the supernatant is better than decanting.
8
Rinse the pellet with 250 µL of 70% EtOH, and cap the tubes.
9
Vortex briefly.
10
Centrifuge for 5 minutes at maximum speed.
11
Without disturbing the pellet, carefully aspirate the supernatant with a pipettor and discard.
12
Dry the pellet in a vacuum centrifuge for 10–15 minutes, or remove the caps and place the tubes in a heat block at 90°C for 1 minute. Note Do not over-dry, or the samples may be difficult to resuspend.
13
Resuspend samples using the appropriate loading solutions for your instrument.
Page 30 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
ABI PRISM™ dRhodamine and ABI PRISM™ Rhodamine Dye Terminator Precipitation Protocols Introduction
We recommend the following protocols for precipitations in MicroAmp 96-well trays, and 1.5 or 0.5 mL microcentrifuge tubes. The same protocols are recommended for dRhodamine and Rhodamine terminator chemistries. The “Shrimp Alkaline Phosphatase (SAP) Digestion Method in MicroAmp Trays” on page 39 and the “SAP Method in Microcentrifuge Tubes” on page 41 are optional. The use of Shrimp Alkaline Phosphatase is a good method for removing all excess dyes.
Ethanol/ Magnesium Chloride (MgCl2) Method in MicroAmp Trays
Reagents and Equipment Required IMPORTANT
♦
Do not use this protocol for BigDye Terminators.
70% non-denatured EtOH/0.5 mM of MgCl2 stored at room temperature.
Note
This reagent can be prepared in situ or as a stock solution.
To prepare the 70% EtOH/0.5 mM MgCl2 stock solution: Step 1
Action Combine the following reagents in a 1.5 mL microcentrifuge tube: ♦
1 mL 70% EtOH
♦
1 µL 0.5 M MgCl2
! WARNING ! CHEMICAL HAZARD. Ethanol is a flammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 2
Vortex briefly to mix.
♦
MicroAmp trays (96-well) and tubes
♦
Strip caps or adhesive aluminum tape (3M Scotch Tape 425-3)
IMPORTANT Other types of aluminum tape have not been tested. Tubes must be sealed completely with strip caps or aluminum tape.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 31 of 42
♦
Variable speed centrifuge with microtiter plate tray holders capable of reaching spin speed of at least 1400 x g (see “Calculating Centrifuge Speed” on page 4.)
EtOH/MgCl2 Method in MicroAmp Trays (dRhod/Rhod): Step
Action
1
Remove the MicroAmp tray from the thermal cycler and the cap from each tube.
2
Add the following reagent to the 20 µL reaction: ♦
74 µL of 70% EtOH/0.5 mM MgCl2
3
Seal the tubes with strip caps or aluminum tape (3M Scotch Tape 425-3), and mix by vortexing or inverting the tray several times.
4
Allow the capped tubes to sit at room temperature for 15 minutes. Note Precipitation times <15 minutes result in loss of short extension products; times >24 hours result in increased precipitation of unincorporated dyes.
5
Place the capped tubes in a tray, and place the tray in the centrifuge.
6
Centrifuge the tray for 30 minutes at the maximum speed (at least 2000 x g, but not greater than 3000 x g). IMPORTANT For spin speeds between 1400—2000 x g, increase the spin time to 45 minutes to ensure good precipitation. Note MicroAmp tubes in MicroAmp trays can withstand 3000 x g for 30 minutes.
7
Remove the tray, and remove the caps or tape without disturbing the pellets.
8
Immediately invert the tray on a paper towel, and place it in the centrifuge again.
9
Spin inverted at 700 x g for 1 minute. IMPORTANT The inverted spin must be performed immediately after centrifugation or loss of signal results. If the tray sits for more than 1 minute before the inverted spin, spin it for an additional 5 minutes at 2000 x g to guarantee recovery of the sample. Removing all residual ethanol during the invert spin is essential. Note
10
It is not necessary to dry the pellet before resuspension.
Resuspend samples using the appropriate loading solutions for your instrument.
Page 32 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Ethanol/ Magnesium Chloride (MgCl2) Method in Microcentrifuge Tubes
Reagents and Equipment Required ♦ Microcentrifuge tubes, 1.5 or 0.5 mL ♦
Microcentrifuge
♦
Pipettor with tips, Gilson Pipetman P200 (Rainin Instruments)
♦
Vacuum centrifuge or heat block at 90°C
♦
70% non-denatured EtOH /0.5 mM MgCl2 at room temperature
♦
70% non-denatured EtOH (ACS reagent grade) at room temperature
Note If precipitations are performed on reduced volume sequencing reactions (10 µL), bring the final volume to 20 µL with deionized water, and then follow the protocol below. If residual dyes are still present, increase the precipitation volume by increasing each component proportionally.
EtOH/MgCl2 Method in Microcentrifuge Tubes (dRhod/Rhod): Step
Action
1
After completion of the sequencing reaction, transfer the extension products (20 µL) to 1.5 or 0.5 mL microcentrifuge tubes.
2
Add 74 µL of 70% EtOH/0.5 mM MgCl2 for a final EtOH concentration of 55%.
! WARNING ! CHEMICAL HAZARD. Ethanol is a flammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3
Cap the tubes, and vortex briefly.
4
Leave the tubes at room temperature for 15 minutes to precipitate the extension products.
5
Place the capped tubes in a microcentrifuge, and mark the orientation of the tubes.
6
Spin the tubes for 20 minutes at maximum speed. IMPORTANT
Perform step 7 immediately after this step.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 33 of 42
EtOH/MgCl2 Method in Microcentrifuge Tubes (dRhod/Rhod): Step 7
Action Without disturbing the pellet, carefully aspirate the supernatant with a pipettor and discard it. IMPORTANT The pellet may or may not be visible. the supernatant must be completely removed because unincorporated dye-labeled terminators are dissolved in the supernatant. The more residual supernatant left in the tube, the more residual terminators are in the sample when it is dried. This results in larger terminator peaks in the sample during electrophoresis. Using a pipettor is a better way of removing all the supernatant than decanting.
8
Rinse the pellet with 250 µL of 70% EtOH, and cap the tubes.
9
Vortex briefly.
10
Centrifuge for 5 minutes at the maximum speed.
11
Without disturbing the pellet, carefully aspirate the supernatant with a pipettor and discard.
12
Dry the pellet in a vacuum centrifuge for 10–15 minutes, or remove the caps and place the tubes in a heat block at 90 ˚C for 1 minute. Note Do not over-dry, or the samples may be difficult to resuspend.
13
Resuspend samples using the appropriate loading solutions for your instrument.
Page 34 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Ethanol/Sodium Acetate (NaOAc) Method in MicroAmp Trays
Reagents and Equipment Required ♦ 95% non-denatured EtOH (ACS reagent grade) at room temperature IMPORTANT 95% EtOH made from absolute EtOH can be inaccurate if the absolute EtOH bottle has pulled atmospheric water. We recommend purchasing commercially prepared 95% ethanol to eliminate variability.
♦
3M NaOAc, pH 4.6 (P/N 400320)
♦
70% non-denatured EtOH at room temperature
♦
MicroAmp trays (96-well) and tubes
♦
Strip caps or adhesive aluminum tape (3M Scotch Tape 425-3)
IMPORTANT Other types of aluminum tape have not been tested. Tubes must be sealed completely with strip caps or aluminum tape.
♦
Variable speed centrifuge with microtiter tray holders capable of reaching spin speed of at least 1400 x g (see “Calculating Centrifuge Speed” on page 4)
EtOH/NaOAc Method in MicroAmp Trays (dRhod/Rhod): Step
Action
1
Remove the MicroAmp tray from the thermal cycler, and remove the cap from each tube.
2
Add the following reagents to the 20 µL reaction: ♦
2.0 µL of 3 M NaOAc, pH 4.6
♦
50 µL of 95% EtOH
! WARNING ! CHEMICAL HAZARD. Ethanol is a flammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3
Seal the tubes with strip caps or aluminum tape (3M Scotch Tape 425-3), and mix by vortexing or inverting the tray several times.
4
Allow the capped tubes to sit at room temperature for 15 minutes. Note Precipitation times <15 minutes result in loss of short extension products; times and >24 hours result in increased precipitation of unincorporated dyes.
5
Place the capped tubes in a tray and the tray in a centrifuge.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 35 of 42
EtOH/NaOAc Method in MicroAmp Trays (dRhod/Rhod): (continued) Step 6
Action Centrifuge the tray for 30 minutes at the maximum speed (at least 2000 x g but not greater than 3000 x g). IMPORTANT For spin speeds between 1400—2000 x g, increase the spin time to 45 minutes to ensure good precipitation. Note MicroAmp tubes in MicroAmp trays can withstand 3000 x g for 30 minutes.
7
Remove the tray from the centrifuge, and remove the caps from the tubes without disturbing the pellets.
8
Immediately invert the tray on a paper towel, and tap lightly to decant the ethanol.
9
Spin inverted at 700 x g for 1 minute. IMPORTANT This inverted spin must be performed immediately after centrifugation or loss of signal results. If the tray sits for more than 1 minute before the inverted spin, spin for an additional 5 minutes at 2000 x g to guarantee recovery of the sample. Removing all residual ethanol during the invert spin is essential. Note
10
It is not necessary to dry the pellet before resuspension.
Resuspend samples using the appropriate loading solutions for your instrument.
Page 36 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Ethanol/Sodium Acetate (NaOAc) Method in Microcentrifuge Tubes
Reagents and Equipment Required ♦ Microcentrifuge tubes, 1.5 or 0.5 mL ♦
Microcentrifuge
♦
Pipettor with tips, Gilson Pipetman P200 (Rainin Instruments)
♦
Vacuum centrifuge or heat block at 90°C
♦
3M NaOAc, pH 4.6 (P/N 400320)
♦
70% non-denatured EtOH at room temperature
♦
95% non-denatured EtOH (ACS reagent grade) at room temperature
IMPORTANT 95% EtOH made from absolute EtOH can be inaccurate if the absolute EtOH bottle has pulled atmospheric water. We recommend purchasing commercially prepared 95% EtOH (ACS reagent grade) to eliminate variability. Note If precipitations are performed on reduced volume sequencing reactions (10 µL), bring the final volume to 20 µL with deionized water, and then follow the protocol below. If residual dyes are still present, increase the precipitation volume by increasing each component proportionally.
EtOH/NaOAc Method in Microcentrifuge Tubes (dRhod/Rhod): Step
Action
1
After completion of the sequencing reaction, transfer the extension products (20 µL) to 1.5 or 0.5 mL microcentrifuge tubes.
2
Add the following to the tubes ♦
2.0 µL of 3 M NaOAc, pH 4.6
♦
50 µL of 95% EtOH at room temperature
! WARNING ! CHEMICAL HAZARD. Ethanol is a flammable chemical and is irritating to the skin, eyes, respiratory system. It can cause nerve and liver damage, CNS depression, nausea, vomiting, and headache. Always work in a fume hood. Obtain a copy of the MSDS from the manufacturer. Wear appropriate protective eyewear, clothing, and gloves. 3
Cap the tubes, and vortex briefly.
4
Leave the tubes at room temperature for 15 minutes to precipitate the extension products.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 37 of 42
EtOH/NaOAc Method in Microcentrifuge Tubes (dRhod/Rhod): Step
Action
5
Place the capped tubes in a microcentrifuge, and mark the orientation of the tubes.
6
Spin the tubes for 20 minutes at maximum speed. IMPORTANT
7
Perform step 7 immediately after this step.
Without disturbing the pellet, carefully aspirate the supernatant with a pipettor and discard it. IMPORTANT The pellet may or may not be visible. The supernatant must be completely removed because unincorporated dye-labeled terminators are dissolved in the supernatant. The more residual supernatant left in the tube, the more residual terminators are in the sample when it is dried. This results in larger terminator peaks in the sample during electrophoresis. Using a pipettor to remove the supernatant is better than decanting.
8
Rinse the pellet with 250 µL of 70% EtOH, and cap the tubes.
9
Vortex briefly.
10
Centrifuge for 5 minutes at the maximum speed.
11
Without disturbing the pellet, carefully aspirate the supernatant with a pipettor and discard.
12
Dry the pellet in a vacuum centrifuge for 10–15 minutes, or remove the caps and place the tubes in a heat block at 90°C for 1 minute. Note Do not over-dry, or the samples may be difficult to resuspend.
13
Resuspend samples using the appropriate loading solutions for your instrument.
Page 38 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
Shrimp Alkaline Phosphatase (SAP) Digestion Method in MicroAmp Trays
Use this protocol for ABI PRISM dRhodamine and ABI PRISM FS rhodamine terminators only. It is a good method for the complete removal of excess dyes. IMPORTANT
Do not use this protocol for BigDye Terminators.
Reagents and Equipment Required ♦
1 U/µL SAP
♦
10X SAP buffer (SAP and 10X SAP buffer available from United States BioChemical together in a kit. Order part number 70103)
♦
70% non-denatured EtOH/0.5 mM MgCl2 stored at room temperature
♦
MicroAmp trays (96-well) and tubes
Note This reagent can be prepared in situ or as a stock solution. See “Ethanol/ Magnesium Chloride (MgCl2) Method in MicroAmp Trays” on page 31 for instructions to prepare a stock solution.
♦
Strip caps or adhesive aluminum tape (3M Scotch Tape 425-3)
IMPORTANT Other types of aluminum tape have not been tested. Tubes must be sealed completely with strip caps or aluminum tape.
♦
Variable speed centrifuge with microtiter plate tray holders capable of reaching spin speed of at least 1400 x g (see “Calculating Centrifuge Speed” on page 4)
SAP Digestion Method in MicroAmp Trays (dRhod & Rhod): Step 1
Action At the end of thermal cycling, add the following reagents to the 20 µL reaction: ♦
2.0 µL of SAP (1 U/µL)
♦
18 µL of 1X SAP buffer (1:10 dilution of 10X SAP buffer)
2
Cap the tubes with strip caps, and incubate at 37 ˚C for 30 minutes.
3
Remove the caps, and add 150 µL of 70% EtOH/0.5mM MgCl2.
4
Seal the tubes with strip caps or aluminum tape (3M Scotch Tape 425-3), and mix by inverting or vortexing the tray several times.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 39 of 42
SAP Digestion Method in MicroAmp Trays (dRhod & Rhod): (continued) Step 5
Action Allow the capped tubes to sit at room temperature for 15 minutes. Note Precipitation times <15 minutes result in loss of short extension products; times >24 hours result in increased precipitation of unincorporated dyes.
6
Place the capped tubes in a tray and the tray in a centrifuge.
7
Centrifuge the tray for 30 minutes at the maximum speed (at least 2000 x g but not greater than 3000 x g). IMPORTANT For spin speeds between 1400—2000 x g, increase the spin time to 45 minutes to ensure good precipitation. Note MicroAmp tubes in MicroAmp trays can withstand 3000 x g for 30 minutes.
8
Remove the tray, and remove the caps or tape without disturbing the pellets.
9
Immediately invert the tray on a paper towel, and place it in the centrifuge again.
10
Spin inverted at 700 x g for 1 minute. IMPORTANT The inverted spin must be performed immediately after centrifugation or loss of signal results. If the tray sits for more than 1 minute before the inverted spin, spin it for an additional 5 minutes at 2000 x g to guarantee recovery of the sample. Removing all residual ethanol during the invert spin is essential. Note
11
It is not necessary to dry the pellet before resuspension.
Resuspend samples using the appropriate loading solutions for your instrument.
Page 40 of 42 User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions
SAP Method in Microcentrifuge Tubes
Use this protocol for ABI PRISM dRhodamine and ABI PRISM FS rhodamine terminators only. IMPORTANT
Do not use for BigDye Terminator chemistries.
Reagents and Equipment Required ♦ 1.5 or 0.5 mL microcentrifuge tubes ♦
1 U/µL SAP
♦
10X SAP buffer (SAP and 10X SAP buffer available from United States BioChemical together in a kit. Order part number 70103)
♦
Microcentrifuge
♦
Pipettor with tips, Gilson Pipetman P200 (Rainin Instruments)
♦
Vacuum centrifuge or heat block at 90°C
♦
70% non-denatured EtOH/0.5 mM MgCl2 stored at room temperature (see “Ethanol/ Magnesium Chloride (MgCl2) Method in MicroAmp Trays” on page 31 for instructions on preparing stock solution)
♦
70% non-denatured EtOH at room temperature
Note If precipitations are performed on reduced volume sequencing reactions (10 µL), bring the final volume to 20 µL with deionized water, and then follow the protocol below. If residual dyes are still present, increase the precipitation volume by increasing each component proportionally.
SAP Digestion Method in Microcentrifuge Tubes (dRhod/Rhod): Step
Action
1
After completion of the sequencing reaction, transfer the extension products (20 µL) to 1.5 or 0.5 mL microcentrifuge tubes.
2
Add the following reagents to the tubes: ♦
2.0 µL of SAP (1U/µL)
♦
18 µL of 1 x SAP buffer (1:10 dilution of 10X SAP buffer)
3
Cap the tubes, and incubate at 37°C for 30 minutes.
4
Remove the caps, and add 150 µL of 70% EtOH/0.5 mM MgCl2.
5
Cap the tubes, and vortex briefly.
6
Leave the tubes at room temperature for 15 minutes to precipitate the extension products.
User Bulletin : Precipitation Methods to Remove Residual Dye Terminators from Sequencing Reactions Page 41 of 42
SAP Digestion Method in Microcentrifuge Tubes (dRhod/Rhod): Step
Action
7
Place the capped tubes in a microcentrifuge, and mark the orientation of the tubes.
8
Spin the tubes for 20 minutes at maximum speed. IMPORTANT
9
Perform step 9 immediately after this step.
Without disturbing the pellet, carefully aspirate the supernatant with a pipettor and discard it. IMPORTANT The pellet may or may not be visible. The supernatant must be completely removed because unincorporated dye-labeled terminators are dissolved in the supernatant. The more residual supernatant left in the tube, the more residual terminators are in the sample when it is dried. This results in larger terminator peaks in the sample during electrophoresis. Using a pipettor to remove the supernatant is better than decanting.
10
Rinse the pellet with 250 µL of 70% EtOH, and cap the tubes.
11
Vortex briefly.
12
Centrifuge for 5 minutes at maximum speed.
13
Dry the pellet in a vacuum centrifuge for 10–15 minutes, or remove the caps and place the tubes in a heat block at 90°C for 1 minute. Note Do not over-dry, or the samples may be difficult to resuspend.
14
Resuspend samples using the appropriate loading solutions for your instrument.
© Copyright 1998, Applied Biosystems ABI PRISM is a registered trademark of Applera Corporation. ABI, ABI PRISM and the ABI PRISM design, Applied Biosystems, BigDye, MicroAmp, and Applied Biosystems are trademarks of The Applera Corporation. AmpliTaq is a registered trademark of Hoffman-LaRoche, Inc. All other trademarks are the sole property of their respective owners. Stock No. 237842-001, P/N 4304655 Rev. A
