PCR & spermidine

[kang at msvax.mssm.edu]

In article <D42IDt.7Iu at news.cis.umn.edu>, horton at cis.umn.edu (Robert Horton) writes:
>Claus B. Jorgensen (cljoh3 at wptemp.kvl.dk) wrote:
>: Hi Bionetters,
>
>: I have a couple of primer pairs that works nicely on the plamid template, but 
>: they fail when I'm turning to genomic DNA. 
>: I have tried different [Mg] and annealing temps, but neither seems to change 
>: the outcome.
>
>: Any suggestions to modifications would be appreciated. 
>
>Genomic DNA is much more complex, in addition to (depending on how much plasmid
>you use) being present in fewer copies. This means the primers are much more
>likely to bind to unrelated sequences and give you "random priming"-like
>artifacts. If you get enough different artifacts, you may not see any bands.
>
>For sure try hot starting your genomic amplifications (i.e., don't add the 
>enzyme 'til the reaction is hot).
>
>You may need longer primers to give enough specificity to amplify from 
>genomic DNA; <~15 bp can work well from a plasmid, while you probably want
>25mers (or longer) for genomic DNA.
>
>No ideas about spermidine...
>
>
>Bob Horton (Ph.D.!)   /\ "Crash programs fail because of the theory that
>U. of Minnesota, CBS  || with nine women pregnant you get a baby a month" 
>1479 Gortner Ave.    /||\   -Werner von Braun.  Disclaimer:"Bob who?"
>St. Paul, MN 55108    ^^   horton at molbio.cbs.umn.edu/(612) 624-3790


Why don't you add DMSO?
My transgenic mice experience tells me that 2% DMSO in your PCR rexn significantly
enhance the quality of your band.
Of course, Changing primer (changing target) would be a good idea. If the
amplified band is less than 800 bp I usually didn't have any problem using
regular Taq and PCR mix recipie from Perkin Elmer.

Chulho Kang
Transgenic mice wizard