what do you call this?... semearing? - (Jun/10/2007 )

Hi friends,

I am doing RAPD fingerprinting on bacteria. Previously it was okay but all PCR results of last week are not clear (see the photo). I didn't change anything. In RAPD the bands should be clear enough to make analysis... but because of the 'smearing' i am not able to do this…by the way is it DNA smearing?

Pls look at the picture and foreword me your advice on how to clean it out

10Q
Doyo

on the gel picture the two lanes at corner are 100bp and 1kbp markers the others are my RAPD PCR products.

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It looks like to me that you have alot of samples loaded in your agarose gel. You have your distinct bands there. Perhaps you can clean it up using some PCR clean up kit (if your sample is PCR) or just normal phenol chloroform method. Hope this helps.

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What's your template like? What differences are there between the four lanes?

Without knowing any details, I'd say your first two lanes have too much template, which has also sheared. Have you had the template DNA for long? have you tried diluting the DNA out to see what happens?

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All lanes were loaded with 10ul of the PCR product, running on 1.5% Agarose at 80V for 1:30hr.
The PCR template amount was 75ng, DNA polymerase 2.5U, primer [50pmol], buffer 2.5, dNTP's 0.2mM, and MgCl 3mM.

DNA was extracted using DNease Qiagen before a month stored at -20oC.

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You do have smearing in all of the wells.
Has the DNA already been cleaned using Qiagen. If its already cleaned up, then the elution solution could be contaminated causing this smear.

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Have you tried using just 5uL on the gel? Too much causes smearing for me.


Amanda

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EVTG is cool now it was bcus of excess enzyme...previously I was using 2.5U but now 1.5unit so no smearing.

Thank you,

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