Big-Dye Terminator Cycle Sequencing and Sequencing on ABI 310
1. After initial PCR and clean up of fragment with Qiagen gel extraction kit (cat. No. 28704) run 5µl on a gel to determine how much to use for sequencing reaction.
Typical amounts sequenced are from 2-5 l from a 30 l DNA sample. The fragment should be very clean and be just visible on the gel.
2. For the sequencing reaction add the following to a PCR tube:
Terminator ready reaction mix 4.0l ( Cat. No. 4303149 - ABI)
DNA template 2-5l
Primer (forward or reverse) 1l
Sterile H2O up to 20l
3. Cycle Sequence on 9600 or 9700 for 25 cycles
Denature 96C for 3 min.
3 step PCR- 96C for 10sec., 50C for 05sec.,60C for 4 min.
Hold at 4C until ready to purify
4. Purify using DyeEx Columns ( Cat. No. 63104-Qiagen)
Follow manufacturers protocols. Do not over dry sample in vacuum.
Resuspend sample in 30l TSR (Cat. No. 401674-ABI) Keep at 4C o/n.
Remember this is light sensitive!!!
5. To run fragment on ABI 310, set instrument up with the following:
- 9ml of 1x genetic analyzer buffer in the buffer reservoir (10x buffer with EDTA- cat. No.402824)
- for sample tube tray prepare position #1- 4ml 1x analyzer buffer
#2- 4ml H2O
#3- 1.5ml tube of H2O
6. Set up capillary (61cm) (cat. No. 402840-ABI) Make sure to use the pink one for sequencing. End of capillary should be just slightly below the electrode. Tape capillary to wall of the machine to prevent movement of capillary. Attach a prelubricated 1ml glass syringe to the pump block.
7. Take out Pop 6 (cat.no. 402837- ABI) and let warm up to room temp. before using. Take approximately 1ml of POP6 using a 5ml plastic syringe and a 20G - 1 inch needle. Take the needle off of the syringe. Attach the syringe with POP6 to the pump block. Fill pump block with POP6 manually. Make sure there are no bubbles in the pump block. Refer to user’s manual for exact filling method.
8. From the 5ml plastic syringe, fill the 1ml glass syringe with POP6. .2ml should be enough for 20-30 samples. The glass syringe should be lubricated slightly with H2O prior to filling in order to prevent friction degradation of the syringe. Make sure there are no bubbles in the glass syringe.
9. Take the plastic syringe with POP6 off of the pump block. Any excess POP6 should be put back into the original vial. Fill another 5ml plastic syringe with water and place onto the pump block. Select the syringe down command from the Function menu. Bring the syringe arm down to just touching the glass syringe
10. Set the ABI 310 temperature to 50C under manual control. Run the seq. fill capillary program under manual control.
10. Set up the sample sheet and the injection list under “File”-new. For the Dye Set/Primer section on the sample sheet, set to Big-Dye (any primer).
The matrix file should be set to “good matrix”. On the injection list bring up the sample sheet and adjust the run time according to fragment length. At this point start denaturing your samples at 95C for 5min.
200bp=40 min
300bp=60 min
400bp=80 min
500bp=100 min
11. Calibrate the autosampler . Set the front and rear alignments so that the capillary is almost touching the silver dot on the autosampler tray. See manual for more detail on how to calibrate.
12. Load the machine with the first sample in the top right hand corner of the autosampler.