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Direct sequencing PCR product


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3 replies to this topic

#1 PostDocTrauma

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Posted 27 July 2011 - 02:18 PM

Hi all

My boss wants us to save money instead of using expensive TOPO kits. I was wondering if anyone has sequenced PCR products from bisulfite treated DNA?

Otherwise, what cloning vectors do you use for bisulfite sequencing?
Thanks

#2 pcrman

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Posted 30 July 2011 - 10:15 PM

You can do direct sequencing of the BSP products. We once made a side by side comparison between direct sequencing and sequencing after cloning (sequencing at least 10 clones), and found results from both methods are pretty consistent. The drawback is that not every PCR product gives you a nice sequencing result, in contrast, sequencing plasmids give you very clean results. Another drawback is that direct sequencing only give you the average methylation level of cpg sites, but not the methylation profile of a single DNA molecule.

#3 methylnick

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Posted 31 July 2011 - 05:44 PM

pGEMT easy works just as well, TOPO is quite expensive.

#4 PostDocTrauma

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Posted 01 August 2011 - 07:21 AM

thanks :)

I would prefer to sequence from clones but cost is now a big issue. Will see how much pGEM is




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