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

[COLLEEN M MCMICHAEL]

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: http://www.med.yale.edu/genetics/ward/tavi/PCR.
html.

Good Luck!

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

>  Message: 3
>  Date: Mon, 29 Jun 2009 19:21:15 -0500
>  From: =?GB2312?B?88O0q8PA?= <zhuchuanmei04 from gmail.com>
>  Subject: [Arabidopsis] Could anyone help me on Arabidopsis genomic DNA
>  	cloning?
>  To: arab-gen from magpie.bio.indiana.edu
>  Message-ID:
>  	<d407ea500906291721v3ef24347m866f1605a9e487f8 from mail.gmail.com>
>  Content-Type: text/plain; charset=ISO-8859-1
>  
>  Hi,
>  I am a graduate student in Washington University. I have trouble in cloning
>  a ~1.9 kb DNA fragment from Arabidopsis genomic DNA recently. The genomic
>  DNA was prepared by CTAB method and was dissolved in H2O.  For 25ul PCR
>  reaction, I use DNA ~500ng, MgCl2 1.5mM, dNTP 160uM, Taq 2.5U, primer 
> each
>  0.2uM, buffer 1x. The PCR program was 94'  5min; 94'  45s, 54' 45s, 72'
>  2min, 20cycle; 72' 5min, 4' for ever.
>  
>  I tried several times, including trying different concentration of DNA,
>  MgCl2... However, I never got the expected 1.9kb band? This was my first
>  time to clone gene from genomic DNA, I have no idea why it turns out 
> to be
>  so difficult. Did you have any suggestions? Thanks in advance.
>  
>  Best,
>  
>  -- 
>  Chuanmei Zhu
>  DBBS(plant biology),
>  Washington University, St.Louis, MO,USA. 63130.
>  
>  Tsinghua U (B.S.)
>  
>  
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>  End of Arab-gen Digest, Vol 50, Issue 15
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