bisulfite PCR: is it my product? - (Mar/20/2008 )
Hi,
I am a student majoy in methylation of DNA. now I 'm quite confused by my experiment. i'm doing bisulfite sequencing and now the PCR products is not what i want.
the sequence in the agrose gel (2%)is not the target seqence, my target sequence is 243bp, but in the gel, it's may be 270bp.(fig1)
but more confused me is that i use 8% PAGE gel run my product, it indicated about 330bp(fig2). i don't know why? is it the product i wanted?
[attachment=4398:2008.03.18_2.jpg]fig1
[attachment=4400:2008.3.15.jpg]fig2
hi
Maybe it's just my dumb luck but i never cared for such differences and it has always been the right choice. It's only gel, don't expect the result to match your expectations in 100%.
First of all think again if your primers are properly designed for converted DNA. Then if your tmplate is human genomic dna you can check your primers again by running genometester (bioinfo.ut.ee/genometester), remember to mark "bisulphite treated genomic dna" - you will see if there are any more sites where your primers could anneal
if all of the above is ok then sequence the product. You can also try restriction analysis before, but then you have to remember that the recognition sites for many enzymes will appear/disappear depending on the methylation status of DNA that you amplified because you will have either "c"s or "T's
I am a student majoy in methylation of DNA. now I 'm quite confused by my experiment. i'm doing bisulfite sequencing and now the PCR products is not what i want.
the sequence in the agrose gel (2%)is not the target seqence, my target sequence is 243bp, but in the gel, it's may be 270bp.(fig1)
but more confused me is that i use 8% PAGE gel run my product, it indicated about 330bp(fig2). i don't know why? is it the product i wanted?
[attachment=4398:2008.03.18_2.jpg]fig1
[attachment=4400:2008.3.15.jpg]fig2
Could the base bias cause the difference in the way the amplicon migrates in the gel? if it is only AT-rich sequence could it run differently from a balanced sequence if you get what i mean??
Nick
Maybe it's just my dumb luck but i never cared for such differences and it has always been the right choice. It's only gel, don't expect the result to match your expectations in 100%.
First of all think again if your primers are properly designed for converted DNA. Then if your tmplate is human genomic dna you can check your primers again by running genometester (bioinfo.ut.ee/genometester), remember to mark "bisulphite treated genomic dna" - you will see if there are any more sites where your primers could anneal
if all of the above is ok then sequence the product. You can also try restriction analysis before, but then you have to remember that the recognition sites for many enzymes will appear/disappear depending on the methylation status of DNA that you amplified because you will have either "c"s or "T's
hi, gangut
thanks, i think the genometester you show me is very useful. but i think there is something wrong with it. when i check my primers, it always show me "the process is running", i wait for 2days, no result. and i have tried many times.
Nick
Hi, methylnick
i'm sure it is AT-rich sequence, but why the "AT-rich sequence" run differently?
i have send the product to sequence, I hope it is what i want.
Hi,
I don't know why you had problems with genometester, i checked it a minute ago and it worked fine, did you click the "check results' button?
AT rich regions can form some secondary structures, loops etc that's why they will run differently