Bisulfite Sequencing Technique - (Aug/02/2006 )
Hi,
I am doing bisulfite sequencing to find methylation status within a certain region of maize (corn) DNA. I have a region of about 1200bps, I have been told before that I should try to fragment my region to no more than 500bps for busilfite treatment to work. Does anyone have any comment that could help me?
Furthermore, I don't know the region outside this sequence to be able to select for certain Restriction enzymes to cut outside my region of interest. Can I just use any RE as long as it is not within my region of interest?
Your help would be greatly appreciated.
>>I am doing bisulfite sequencing to find methylation status within a certain region of maize (corn) DNA. I have a region of about 1200bps, I have been told before that I should try to fragment my region to no more than 500bps for busilfite treatment to work. Does anyone have any comment that could help me?
Yes, that's right. the shorter the amplicon, the easier the amplification.
>>Furthermore, I don't know the region outside this sequence to be able to select for certain Restriction enzymes to cut outside my region of interest. Can I just use any RE as long as it is not within my region of interest?
You can cut with any enzyme that don't cut within your amplicon. Alternatively, you can shear your DNA with a syringe and a narrow gauged needle by passing the DNA through the needle several times.
Good luck.
you could also try shearing your DNA with a 21G needle, and this would help reduce the complexity and randomly shear your DNA.
Nick
is it ok to shear DNA with a needle after bisulfite conversion or it has to be done before?
It is done before bisulfite modification to facilitate DNA denature
Dear pcrman, Thanks so much for your response.
Also, I am a bit confused about the need to do 20 colonies or 10 for that matter. If you are selecting for the white colonies doesn't that already tell you that you have the DNA inserted?
Your help would be greatly appreciated,
Kam
QUOTE(pcrman @ Jan 11 2005, 03:46 PM)
After you have done a sucssessful PCR, purify your PCR products and use the fresh products to do a TA cloning (ligation->transformation->plating). You will see white and blue colonies on the plates. For each PCR reaction (transformation) or DNA sample, randomly pick ten white, big, shiny and well isolated colonies using a yellow pipet tip and drop the tip into a 14-ml or 50-ml tube containing 2-5 ml LB medium with antibiotics added. Grow the bacteria overnight at 37C with virgorous shaking. The next morning, take out the tubes and take 1 ml overnight growth for miniprep. You can pick more than 10 colonies (such as 12) just in case some minipreps fail. If cost and labor is not a problem, screen 20 colonies. After you got the plasmid DNA, send it out for sequencing.
Hope that helps.
Selecting 10-20 colonies is for sequencing, which means you have to sequence 10-20 colonies (white) to get a good representation how many DNA molecules are methylated and how many are not because you start with a mixture of potentially methylated and unmethylated DNA molecules. The number 10 or 20 are just arbitrary ones. Certainly you can sequence all white colonies if money and labor is not a problem.
Thanks Mr. pcrman,
I see, and with the selection of the white colonies there is no way of telling if the DNA is methylate or not. So once the sequencing results return I would look at all the data and conclude. So then there is a possiblity that the sequencing results could come back with all 20 or more colones being unmethylated just by chance? correct?
Cheers, K.
you could cut with methylation specific RE to see if it was methylated.
general test, but since plants have heavily methylated cpg's throughout, you will get a lot of activity.