Problems in DNA sequencing after gel purification - (Jul/04/2006 )

dear Colleagues:
I cant optimise my PCR reaction to get just one specific band so I have to purify the needed band with gel extraction kit after I do it and send them for sequencing I get no results and they canot sequence my sample but when I send other bands that are spesific and are not purified by gel extraction kits (they are PCR products) I dont have any problem sequencing them.
what I wonder is what is the reason and what can happen durring the gel extraction that make my samples unsequencable?
thanks

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It sounds as if your gel extraction procedure is introducing a contaminant. You could try gel isolating a band you know has been sequenced well directly from a PCR reaction for comparison, to make sure it isn't the band. How are you gel purifying? Are you exposing the gel to short wave UV before extraction? What buffer are you eluting the DNA in? Do you measure the DNA concentration after gel extraction?

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Alcool traces is a potent inhibitor of polymerase and disturb very much sequencing ! Some chaotropic salts also inhibits very much reaction if not perfectly removed !

Pesji

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try to reamplify the gel elute band
good luck
shashi

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Hai,
I had the same problem too, i figured it out....i used Qiagen Gel Purification kit, the only extra step we need to do is to dry the sample after eluting your purified sample at room temperature for atleast two or three hrs before you use the sample for sequencing, by doing so i think the ethanol in your purified sample evoporates and this sample when sequenced gives good results, try it out, hope this helps.

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I use the Qiagen gel purification kit. I leave the wash buffer for 5 min. and wash 2-3 times and then elute it. Usually the DNA is quite pure.

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I extract the DNA with Core one kit and the buffer is Tris HCL 5mM but the first elution buffer is provides by the kit and I dont know the exact component of it , befor cutting the gel I should put the gel on uv to understand where the band is , I dont measure the DNA with sepectorophotometr but I run it on 1.5% agarose gel and I see the presence of that band and its very clear and sharp but I dont know why they cant sequence it:(

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What is the pH of the Tris ? If it's too acidic it might interfere ! Does it include also some EDTA in the elution buffer or just Tris ?

Pesji

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thanks for your attention
ph of Tris HCl is 8 and its pure no EDTA is added
and I found out that the washing buffer is ethanol added

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Hello

I have the same problem with purifying my products and I was just wondering what should one do to avoid the alcohol/chaotropic salts from inhibiting the sequencing reaction?

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