A sleazy way to avoid direct PCR Sequencing
findley at u.washington.edu
Thu Jan 20 14:13:29 EST 1994
Everybody knows that sequencing a single subclone from a PCR reaction
isn't too smart because of Taq-mediated mutagenesis.
And direct sequencing of PCR products can be a technical challenge...
Can anybody tell me what might be wrong with the following technique
which I used recently to sequence something I PCR'd?
In order to increase the complexity of the template I wanted to
sequence, thereby avoiding individual artefacts I did this:
After cleaning up my PCR reaction and polishing the ends (I have a very
efficient protocol for this) I subcloned the fragment into a very well
phosphatased (blunt) vector and transformed some E. coli.
Now, rather than pick ONE colony for sequencing, I scraped about one
*thousand* colonies off the plate and resuspended and mixed (well) the mass
in a small volume of medium. Then I used a bit to inoculate an overnight
with. I did an Acid-Phenol miniprep for sequencing and got nice clean
sequence. (I used an internal primer, so any vector subclones wouldn't
be primed by my internal primer).
In this way, I was able to sequence many different PCR products with
Now I know this looks pretty retrograde, but for somebody who might not
be able (for whatever reason) to sequence PCR products directly:
Is a thousand colonies enough to be pretty sure of the sequence?
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