DNA sequencing protocol
Jason W. Lilly (7-1-99)
1.) Start with fresh plasmid DNA purified with Qiagen plasmid kits.
2.) Resupsend DNA in H20 only.
3.) Adjust concentration to 250 ng/uL.
4.) Set up primers to a concentration of ~ 2-4 pM/uL.
5.) Set up reaction as follows:
4 uL H20
4 uL 2.5X Buffer
4 uL DNA
4 uL Big-Dye Enzyme Mix
4 uL Sequencing Primer
6.) Run on PCR machine using following conditions:
95 C for 2:30 minutes
96 C for 20 sec
50 C for 20 sec (adjust annealing
temp for your primers) (this is optimal for pUC sequencing primers)
60 C for 5:00 minutes
50 cycles
4 deg. C HOLD
7.) Clean PCR reactions with Qiagen DyeEX removal kit.
Alternatively, ethanol precipitation
is acceptable.
50ul 95% EtOH + 2.1 ul 3M Na0Ac
pH 4.6 add 20ul of PCR reaction, mix well let sit on bench.
5 mintues at room tempeature spin
at max for 20 minutes decant.
Wash pellet with 70% EtOH, spin
at max for 15 minutes.
8.) Speed-vac reactions to dry.
9.) Take to Biotech center for sequencing (Expect a two day wait).
NOTE: If you have lower DNA concentrations adjust primer accordingly (ex. 200ng of PCR product you 3-5 pM of primer).