shorter PCR products after bisulfite treatment and cloning - (Jan/12/2006 )
i treat genomic dna with bisulfite, then i do pcr for bisulfite sequencing. when i run a gel i can see just my band. the problem is that after cloning (TOPO TA) when i check my clones, either restriction enzyme digestion or PCR with M13 primers, i don´t have any right clones (0 out of 16, the last time i tried). i have also tried to to cut the band from the gel and purify it before doing PCR, but it is (surprisingly) just the same: i always have shorter products than expected. any ideas?
thanks a lot
you TOPO TA reagents could be contaminated with some PCR amplicon that is not yours. Try using a fresh kit.
What is the size of your expected insert and how short is the actual insert?
If the wrong insert is only slightly shorter than the right one, the problem could be that non-specific amplification of similar size as the expected product was not separated well during electrophoresis.
hi, thanks for answering!
i don´t think the TOPO TA is contaminated because in that case, it would be contaminated with a pool of pcr products, because in each clone i obtain a different band. i don´t think either that it is a problem of cutting a close band, because my band is 350bp and i obtain bands from 100bp or less to 400bp. apart from that, i find very curious that even when the correct band is the only band i can see in the gel, i can´t get a single correct clone. i could understand some non-specific products when cutting the band, as pcrman says, but only getting non-specific products...
I have had contamination in a pGEMT stock and indeed there were variable sized bands, a PhD student somehow contaminated it, we threw that away and used a fresh stock from which the problem went away.
out of interest, what enzyme are you using to cut the insert, and could there be a site within your insert that the enzyme is cutting at, also if you have bisulfite converted it is possible that a new site is generated for the enzyme you are using.....
hi, thanks again for your interest!
i´m cutting with EcoRI, but i have already checked and there are not any sites/new sites in the insert. besides, i obtain the same result doing pcr instead of cutting.
yes, maybe it´s a contamination problem. i will change stocks.
thanks a lot