PCR sequencing

chai_z at wehi.edu.au chai_z at wehi.edu.au
Mon Jan 18 20:20:13 EST 1993

In article <1993Jan16.183926.14051 at galileo.cc.rochester.edu>, DSCT at db1.cc.rochester.edu (DAVID SCOTT) writes:
> I would like to know if anyone has any experience going from cDNA to sequence.
> I am currently (the gel is running now) doing the following.
> 1. Generated cDNA with specific 3'end of gene primer.
> 2. 20 rounds of PCR with both primers
> 3. purification of PCR product. (geneclean)

Normally I do not purify the DNA if I got only one band. It is critical to get
rid of the free primers present in your PCR reaction. You may use Promega 
Saphacryl (S200 or S300 or S400) spun column, or Magic PCR Preps, or other
Primer Erase columns to separate your DNA products from the small molecules
including the primers (very quick and convinient).

> 4. addition of both primers ( the 5' of which has been end labled with 32P )

For sequencing reaction, ONLY ONE primer is needed in a reaction. You may set 
two sets of reaction using one of the two primers for each reaction so that you
 may get sequences from both ends.

> 5. PCR in 4 tubes with the different dNTP's and ddNTP's(diffent ddNTP in 
>              each tube). PCR done for 20 cycles. 

You may have noticed that the proportion of each ddNTP in the reaction is not
exactly the same. I normally did PCR sequencing for 30-40 cycles, depending on
the amount of your template DNA and the efficiency of primer binding
(temperature, etc)

> 6. I then loaded 3microliters of each reaction onto the sequenceing gel (8%)
> 7. I don't know why it won't work, but I have a feeling I am missing something.

Have you gotten a smear or what?
> Any help/experience with doing this would be apreciated.
> 					Gavin Fischer
> 					University of Rochester Cancer Center
> either post or e-mail to dsct at db1.cc.rochester.edu
> 		         dsct at uordbv 
> 	Thank you

I use New England Lab's CircumVent kit for sequencing reactions. I found the
ratio of DNA and Primer (~300ng/2pmoles) was important for a clear reading. The
 best I've gotten is over 400 bases clear reading (two runs of electrophoresis).

I hope this helps.

Zhonglin Chai

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