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Old 03-22-2011, 06:39 AM   #1
majo82
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Default RRBS library prep.

Hi all!
I tried to prepaare a library for genome wide methylation analysis with RRBS.
Unfortunatly I saw that MspI is a very very annoing enzyme which digest very slowly and unefficiently. The Meissner paper which reported this tecnique said that they use 5-20 U of enzyme for 30 ng-1 micrograms (nat methods 2010) or 10-100 U of enzyme for 1-10 micrograms (nature 2008) of genomic DNA. Here in institute, to obtain a "smear" of digested DNA (visible only upper 700 bp) we use 100-300 U for 1 microgram of DNA and digested overnight. We tried a lot of things ad the best seems this, far from conditions disclaimend in the paper..Is there someone who can suggest me somenthing new?
I load into gel 1 micrograms of digested dna and purified the "gel zone" (when i never had seen any smear) 100-400 bp and I obtained only 1 ng of DNA!!!!!
And if I would digest twice the dna (that is digest and re-digest?)
please help me
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Old 03-23-2011, 09:56 AM   #2
volks
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keep in mind that you are enriching for ~1% of the original genome. so starting from 1ug you would get out 10ng at the max if you dont overdigest.

edit: this estimation is for human, fragments ranging 40 to 240bp.

Last edited by volks; 03-23-2011 at 10:00 AM.
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Old 03-28-2011, 07:13 AM   #3
majo82
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Thank you volks!
I think that anyway is better to load my digested DNA after the adpter ligation for library preparation...And use polyacrilamide gradient gel for checking the digestion.
I write to Meissner (who developed the technique) and Bock and they gave me the newest protocol paper from Meissner lab (with pictures of the gels)
http://www.nature.com/nprot/journal/....2010.190.html

I think this should improve the yield...
Thank you anyway
ale
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Old 03-28-2011, 07:43 AM   #4
C.R.
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I think the trick is to use an appropriate gel for checking. On a high percentage gel the DNA looks uncut, but on lower percentage gel my samples look similar to the the example from the Zymo web page: http://www.zymoresearch.com/zrc/img/product/D5014.jpg
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Old 03-29-2011, 11:41 PM   #5
majo82
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What type of gel do you prepare?I obtain a modest "smear" with agarose 1.%...
thanks
ale
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Old 04-01-2011, 12:32 AM   #6
C.R.
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I digest 1g DNA and use a standard 0.7% agarose gel. I see a smear starting from uncut = approx. 10,000 to 100-200. The intensity below 200 bps gets lower. With 1% it should already look much better than with 2%.
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