A beginner in RACE PCR, or possibly inverse PCR needs advice!

paiks at gunet.georgetown.edu paiks at gunet.georgetown.edu
Mon Mar 6 15:02:54 EST 1995


>   mbxrh at unicorn.ccc.nottingham.ac.uk (Richard Hastings) writes:
>  I know the principle
>  behind the two techniques, what i need to know is how well they work,
>  the advantages of each, any problems you've had with them had and how
>  you got around them.
>  Any help i get would be greatly appreciated!

>  
>>>>you seem to be trying to get 5' flanking genomic DNA sequence. In that
case, a anchor mediated PCR cloning kit from Takara (available from Pan Vera) 
works really well. I used this kit to clone 5' flanking genomic DNA for my 
proviral insert and successful in every attempt so far.
If you want to try inverse PCR, it would not be a bad idea to do a southern
after recircularization of digested DNA with the oligo, cut out the band, then do PCR.
 For 5' RACE, Gibco-BRL's kit worked very well for me, if I used 
nested priming. 
I do not have any experience with it, bit I know that many netters found
Clontech RACE kit frustrating.
good luck,
Soon Paik, M.D.
Lombardi cancer Center
paiks at gunet.geogetown.edu




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