DNA migrate differently in agarose vs PAGE gel - (Aug/15/2011 )
i am currently working a gene in leukemia. The mutant allele has been sequenced to contain 527 base pair. when i run an agarose gel to confirm, it did appear in the position approximately equal to 527 base pair according to the DNA ladder. HOWEVER, when i run a PAGE, it shows up at a different poistion!!(it is at about 600 base pair in PAGE!!!!). Yet the conditions were the same when i run the PAGE and agarose gel (150v, 30 mins)
I just wonder why this thing will happen and is it possible to happen.
Thx for your help!!
Many things can affect DNA migration in gel. Check this page "Top 10 Fun Facts for DNA Electrophoresis" to see if there is anything that might have caused the abnormal migration. For example "On a polyacrylamide gel, DNA fragments having AT-rich regions migrate slower than other DNA fragments of the same size."
According to Fermentas troubleshooting, here is a list of things that may affect migration of DNA in gel:
Atypical migration due to different DNA sequence or structure. During high resolution electrophoresis DNA fragments of equal size can migrate differently due to differences
in DNA sequences. AT rich DNA may migrate slower than an equivalent size GC rich DNA fragment. DNA structures such as nicked, supercoiled or dimeric molecules will always show different mobility on gels compared to an equivalent DNA size standard.
Gel shift effect. The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes may alter DNA migration in the gel or cause the DNA to remain in the gel wells. High salt concentration in the sample may also cause gel shift effects.
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PCRman, I suggest you should pin this post. the link you provided is quite interesting...
Many thx to pcrman.
But actually i am dealing with the same gene migrate differently in different gels instaed of different gene of the same size miigrate differently in different gels. So, would u think the difference in base pairs observed in different gel of the same gene be possible?
Maybe. No matter it is agarose gel or PAGE gel, you are dealing with relative migration of your DNA and the ladder.