PCR & spermidine
Dr Keith Anthony Grimaldi
regokag at ucl.ac.uk
Sun Mar 5 12:27:27 EST 1995
kang at msvax.mssm.edu writes:
>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.
I have also had this problem particularly with GC rich regions. I agree with
Hot-Start and DMSO (up to 10%), these have helped in some cases. You could
also try formamide (up tp 5%). Spermidine for me has not helped but that is no
reason not to try it. Another possibility is a 5 or 10 min denaturation step
at 99 degrees before PCR, or boil the genomic DNA before adding it to the mix.
Or denature with fresh NaOH (0.4M) ppt then PCR. Something might work although
in some cases the nothing has worked execpt getting a new primer, even one
only 10 or 20 bases removed from the original. I haven't had much joy with
programs which claim to choose the best primers. It seems to me that the only
way is empirical.
Best of Luck
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