Cloning bisulphite converted amplicon - Need help on getting more clones (Dec/03/2008 )

Hi All,

I'm trying to clone a 550bp bisulphite converted amplicon (most or all Cs are now Ts), but i seem to get very low cloning efficiency. The vector yeilds are also very low (50 to 100ng/ul compared to 500ng/ul usual yeild with same techniques). I have tried cloning with pGemTeasy and DH10B and JM109 cells. I have also tried a TOPO kit and the efficiency is still low.

Can anyone suggest any thing i can try to get the cloning efficiency higher?
Has anyone tried cloning a bisulphite converted amplicon of this size?

Thanks

BioNoob

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I have not seen any thing special for TA cloning of PCR products from bisulfite modified DNA compared to regular TA cloning. Some things you may want to check include:
Are your PCR products freshly made at ligation? What vector:insert ratio? How is your positive control (intact plasmid)? How did you purify your PCR products?

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Insert to vector ratio has been about 3:1 for most ligations. The ligations have been performed up to 2 months after isolation, but usually within the first week after isolation. I am using a Qiagen spin column kit to purify from 2% agarose. The last isolations were quite low concentration, so i'm going to phenol/chloroform isolate and resuspend in 1/3 volume. In the past this hasn't increased the transformation efficiency though.

Do you have any ideas on the amplicon size? Is 550bp too large perhaps? (cells don't seem to be happy amplifying it). What size are most bisulphite amplicons?

Thanks

810N008

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You should do the ligation immediately after PCR and purification. Although you can store the PCR product for a week or so, before purification I would add a bit Taq polymerase and do a extension step for 5 min.

As long as you can amplify 500 bp products, TA cloning is not a problem with that size.

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Will do...

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