Sequencing with Vent

This is a good method that works well with any quality DNA. The ratio of DNA to oligo is important (see booklet). It is particularly useful when sequencing several DNA's with the same oligo and less so when sequencing one DNA with many oligos. It is simpler to set up that sequenase (no denaturing or annealing) but is a pain to load the gels behind a screen. This method gives sequence next to the oligo but does not read as far as sequenase (~150bp on a short run. Run short-short runs.)

1) Set gels

 

2) PNK Reaction

H20 19.5l

PNK buffer 2.5l

10.5 pMol primer 1.0l

32P-ATP 1.0l

PNK 1.0l

Incubate for 30mins at 37oC

 

3) Add 3l of each dideoxy to color coded PCR tubes keep on ice.

 

4) Make the following premix:

1X 5X 10X

8.0 40 80 H2O

1.5 7.5 15 Vent buffer

1.0 5.0 10 Triton 30X

1.0 5.0 5.0 Vent enzyme

Add whichever of the following are constant to the premix, then redistribute and add the other item individually to create 15l premixes for each sample to be sequenced.

1.0l DNA/reaction (1.0lif miniprep/ 50 ng if plasmid prep/ 1.0l if PCR fragment)

2.5l Labelled Primer from step 1

Remember once the oligo is added the rxn is hot and must be handled behind a screen.

5) Add 3.2l of the premix to each of the four dideoxy's.

Add 1 drop of oil and perform 20 cycles of PCR 1/1/1at 94/56/72 oC

 

6) Add stop soln and heat samples

 

7) Pipette samples onto parafilm to remove oil using a standard yellow tip. Then repipette onto the gel using a gel tip. These steps must ll be done behind a screen.