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Will glycogen affect the CT conversion reaction


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#1 freeda

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Posted 17 March 2009 - 05:57 PM

Hi, everybody.
I have some problems with my bisulfite sequencing. Afer restriction endonuclease digestion and proteiase K treatment of my genomic DNA (about 1ug), I purifed the DNA with phenol chloroform. But I found it was difficult to see the pellet afer washing with 70% EtOH. Can I add some glycogen to help precipitation? Will the glycogen affect the conversion rate of unmethylated C to T? I used the EZ-DNA methylation Gold kit, and the conversion procedure was the following: 98℃ for 10 minutes, 64 ℃ for 2.5h and 4℃ for 5h. But I can't get full conversion. I hope somebody can help me solve the problem. Thank you very much

#2 pcrman

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Posted 17 March 2009 - 09:47 PM

You can add glycogen to help DNA precipitation. To my best knowledge, it won't affect DNA conversion. Previously I used glycogen to precipitate DNA from microdissected samples and did not find it a problem.

#3 freeda

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Posted 17 March 2009 - 11:20 PM

I used the EZ-DNA methylation Gold kit, and the conversion procedure was the following: 98℃ for 10 minutes, 64 ℃ for 2.5h and 4℃ for 5h.
After TA cloning, I have sequenced 5 clones. And 2 clones were converted completely, but in 3 clones, only 15%-25% of unmethylated Cs were converted to T.
So how could I improve the conversion rate? Could I operate as the following: 98 ℃ for 10min, 53 ℃ for 30min, 12 cycles of 53 ℃ 6min and 37 ℃ 30min, and during the cycles, insert 95 ℃ 1min for every 3 cycles. At last hold 4 ℃ for 5h

#4 methylnick

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Posted 18 March 2009 - 03:27 AM

may boil down to your assay/primer design in that they are not specifically amplifying fully converted templates?

Nick

#5 freeda

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Posted 18 March 2009 - 05:21 AM

may boil down to your assay/primer design in that they are not specifically amplifying fully converted templates?

Nick

I designed the primers within the region that contained low Cs, and it must direct the amplification of unmethylated templates. In addition, I just treated the plasmid, so theoretically all the Cs should be converted to Ts.
I'd like to know if the reaction conditions I mentioned above will work well.

#6 Davo

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Posted 22 March 2009 - 02:31 PM

Your primers need to avoid CpG-cytosines, but actually want a few non-CpG-cytosines in there. I'm not sure if thats what you mean when you say 'contained low C's'.

I can't comment on the reaction conditions but the Qiagen Epitect kit uses a few cycles of denaturation and then lowers to 50 or so degrees. You should be able to find the product manual on the net if you want to look it up.

#7 methylnick

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Posted 23 March 2009 - 01:51 AM

Freeda,

you are following the zymo kit's to the T, so the reaction conditions look good.

I am a bit concerned about the primer design, if you are picking primers with low C's this would mean that there are very few conversion events and therefore you primers are not really targeting converted template as distinct from unmethylated template.

You said you are only treating plasmid DNA so I can assume you are looking at a mammalian sequence that you have inserted into a plasmid? Because you could be looking at non-CpG methylation if you are not working with mammals.

cheers

nick

#8 RKA

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Posted 21 April 2009 - 12:52 AM

Dear All,

I need some help working with EZ DNA methylation kit. I wanted to bisulfite convert the FFPE material to study methyaltion status further.
So i have tried the EZ methylation start up kit, which also provided with the universal methylated genomic DNA and control primers hMLH1, hMLH2 to check for the convertion whether it worked or not.
After the bisulfite convertion i have done the control PCR reaction with the above mentioned primers (which should yield a product of 182 bp..if the convertion is complete), but i could only see the product formation in the positve control but not with my FFPE material ??? I have checked the quality of the FFPE DNA using oligo's pairs not related to a mehylated related regions that yileds a 160 bp product and the amplification worked on FFPE material and i could see the product.
Its really an essential step to confirm the bisulfite convertion to proceed further to avoid false positive results. Can someone help me how can i confirm the bisulfite convertion ??? is there any thing which i can change in my PCR conditions etc...i have used 2 Ál of bisulfite converted DNA for the control PCR reaction and the reaction as follows, 95 for 10 min, 94.5 for 30 sec, 59 for 30 sec, 72 for 60 sec, 72 for 7 min, 35 to 40 cycles...4 degree hold..
FOr the bisulfite convertion...as mentioned in the protocol i used...98 for 8 min, 64 for 3.5 hours, 4 degree hold.

The positive control worked fine??? (this confirms the bisulfite convertion) y not in FFPE DNA ??? ..I have used the unconverted DNA as negative control and ofcourse i could not see any product with that ...Your suggestions are appreciated...Thanks and regards, Raj

#9 Bionewbie

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Posted 26 April 2009 - 05:13 PM

If the positive control worked fine, it may be the primers you have designed. Keep in mind that when designing primers for bisulfite converted DNA that you need to treat the converted cytosines as thymines.




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