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Reply to "Could anyone help me on Arabidopsis genomic DNA" (Arab-genDigest, Vol 50, Issue 15)

Reply to "Could anyone help me on Arabidopsis genomic DNA" (Arab-genDigest, Vol 50, Issue 15) - Arabidopsis and Plant Biology

Reply to "Could anyone help me on Arabidopsis genomic DNA" (Arab-genDigest, Vol 50, Issue 15) - Discuss Arabidopsis and Plant Biology Research.


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Old 07-01-2009, 03:10 PM
COLLEEN M MCMICHAEL
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Default Reply to "Could anyone help me on Arabidopsis genomic DNA" (Arab-genDigest, Vol 50, Issue 15)



Hi Chunmei,

I have often had problems with PCR using genomic DNA as a template. For the most part, using the CTAB DNA preparation versus other faster, yet dirtier, preparations has helped tremendously. Since you are already using this preparation method, you might now want to try using additives that will help melt your DNA/primers, stabilize the polymerase, etc (for example add DMSO, glycerol, glycine betaine or BSA). BSA has helped more than any other additive for me. Also, increasing the buffer (complete with Mg2+) concentration can also help (up to 1.6 X final). Finally, have you tried varying the annealing temperature for your primer set? I suggest using a gradient machine to test a rangeof tmperatures. Please keep in mind that all PCR products are unique in their needs, and that the best conditions for your reaction must be determined empirically. Here is a link to a website that helped guide me toward possible PCR variations to try: [Only registered users see links. ].
html.

Good Luck!

Colleen McMichael
Bednarek Lab
219 Biochemistry Addition
(608) 263-0314 x3237


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