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b612's Content

There have been 5 items by b612 (Search limited from 18-July 18)


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#92800 X-inactivation chromosome

Posted by b612 on 21 November 2010 - 02:27 AM in DNA Methylation and Epigenetics

Hi everybody!!
I am interested to know how I can differentiate between active X-chromosome and inactive X chromosome.
Thank you in advance!!!



#90909 isolate DNA from amniotic fluid

Posted by b612 on 29 October 2010 - 11:32 PM in Molecular Biology

Hi!!!
I need a method for isolating DNA from amniotic fluid for microarray assay.
I used QIAGEN KIT but i didn´t get enough concentration (<100ng/microL)
Do you Know any good protocol???



#90908 Problem with Qiagen EpiTect Bisulfite Kit

Posted by b612 on 29 October 2010 - 11:22 PM in DNA Methylation and Epigenetics

are you expecting your samples to be unmethylated or methylated? does sound like your conversion has worked.

Nick



Thank you! yesterday i had good results!!!!!! the problem was in methylation assay.



#90540 Problem with Qiagen EpiTect Bisulfite Kit

Posted by b612 on 26 October 2010 - 11:01 AM in DNA Methylation and Epigenetics

Hi!
I am using Qiagen EpiTect Bisulfite Kit for detect methylation status.
I isolated DNA, quantified it, and I did PCR assay.
I have only amplification in PCR with primers for DNA methylated.
I have not amplification in PCR with primers for DNA no methylated.
I think the problem is in bisulfite treatment.
Please!!!
Help!!!
Where is the problem???



#90407 methylation specific PCR assay (PWS/A syndrome)

Posted by b612 on 25 October 2010 - 04:41 AM in PCR, RT-PCR and Real-Time PCR

Hi everybody!!!
I have a big problem!!!! I want to do diagnostic of Prader-Willi/Angelman Syndrome (uniparenteral disomy). I need to know the methylation status. For this I use BISULFITE KIT (Qiagen). Then I have quantified DNA concentration of my samples. Then I have to do methylation specific PCR assay. I have next reactives for each sample:

Buffer 10x= 3 microL
MgCl2 25Mm=2.4microL
Taq Gold 5U/microL=0.6microL.
dNTP ´s 2.5Mm=2.4microL
Primers (maternal primer, paternal primer) (100microM each)= 1.5microL
DNA 30ng/microL.=1.5microL
Water=18.6microL
Total volumen= 30microL.

I use program of PCR from a Kubota´s article “METHYLATION-SPECIFIC PCR SIMPLIFIES IMPRINTING ANALISYS”: 95ºC, 10´, 35 cycles: 94ºC 30”, 62ºC 30”, 72ºC 30”, and final extension 72ºC 10´.

Then I have separated PCR products in 3% agarose gel.

In this moment I have got amplificated only in samples treated with maternal primer.

Somebody knows Where is the problem!!???!!!
, PLEASE!!!

Thank you in advance!




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