Problem cloning bisulfite PCR BSP product - (Nov/16/2009 )
Hello,
I'm trying to investigate the methylation status of the promoter. I do the bisulfite modification of genomic DNA using Invitrogen kit, then I succesfully amlify my regions of interest by PCR. I use Taq polymerase in PCR reaction to generate 3' A ends of PCR product. I clone PCR products using TA method into pTZ57R/T. Then transform cells and plate them on ampicilin and IPTG/X-gal medium.
The problem is, that most colonies I get have insert of 100-200 bp less than needed. And it is the most common result. I try checking them by using both colony pcr (M13 primers) and restriction analysis (PvuII) and I get consistently bad results.
Generally I got some plasmids with correct insert and succeeded sequencing them, so that shows my work method is generally right.
But the sucess ratio is unexpectedly low - about 1 out of 100 tested. I checked my plasmid stock. It is not contaminated.
Anyone has any ideas what is going on ? If you can't give any suggestions how to succed, at least write some theoretical considerations about what is happening there.
thanks.
How large is your PCR product? Large amplicons (>500 bp) won't amplify well in bisulphite PCR.
What do you mean when you say you get 'bad results' from the colony PCR? Did you sequence the shorter insert to see what it is?
Mushin on Nov 18 2009, 06:41 PM said:
What do you mean when you say you get 'bad results' from the colony PCR? Did you sequence the shorter insert to see what it is?
I'm generating various amplicons from 200 to 400 bp. I ligate them into plasmid, transform E. coli, choose colonies with inserts (blue/white selection) and then do colony PCR with M13 primers followed by agarose gel electrophoresis, to see if the plasmid has the insert of the needed size, before sequencing. And mostly, the insert in the plasmid is 100-200 bp shorter than expected.
Really appreciate your insights !
how do you clean your amplicons?
you could be just cloning primer dimer!
methylnick on Nov 24 2009, 10:58 PM said:
you could be just cloning primer dimer!
There is hardly any possibility of that. I used two ways: cutting the amplicon from agarose gel and then purify using spin column, purify with the spin column without electrophoresis. I get the same results wit both of these ways. Also the fragmens inserted are too big to be primer dimers ~100-150 bp.
Can it be possible that bisulfite modified DNA is somehow unstable and get fragmented? I use nuclease free water everywhere.
Jeronimas on Nov 16 2009, 12:24 PM said:
I'm trying to investigate the methylation status of the promoter. I do the bisulfite modification of genomic DNA using Invitrogen kit, then I succesfully amlify my regions of interest by PCR. I use Taq polymerase in PCR reaction to generate 3' A ends of PCR product. I clone PCR products using TA method into pTZ57R/T. Then transform cells and plate them on ampicilin and IPTG/X-gal medium.
The problem is, that most colonies I get have insert of 100-200 bp less than needed. And it is the most common result. I try checking them by using both colony pcr (M13 primers) and restriction analysis (PvuII) and I get consistently bad results.
Generally I got some plasmids with correct insert and succeeded sequencing them, so that shows my work method is generally right.
But the sucess ratio is unexpectedly low - about 1 out of 100 tested. I checked my plasmid stock. It is not contaminated.
Anyone has any ideas what is going on ? If you can't give any suggestions how to succed, at least write some theoretical considerations about what is happening there.
thanks.
Does anyone has some ideas how to solve that ?
Thank you.
Hi there,
I realise that this reply is pretty late now but I do have one suggestion! Bisulphite conversion can often introduce large streches of poly A and poly T into your PCR products. These types of regions can be problematic for Bacteria during replication of the plasmid and can cause rearrangments which could generate your shorter products. You could always try and change the strain of E. coli you are using to propagate your transformed plasmids. For instance if you are using DH5 try switching to XL-1, this may help to overcome your problem as the plasmid may be more stable in one strain than another. Also, particular genotypes of bacteria are better at coping with repeptive regions than others, check the genotype of your specific strain and check that it is RecA-
Hope that helps,
Jon