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Problems with BSP using Methprimer


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#1 aurelie

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Posted 10 December 2004 - 05:43 AM

Genomic DNA has been converted using EZ methylation kit. Then , primers for BSP have been designed using Methprimer software on different regions.

We have different controls of methylation : controls unmethylated, controls 100 % methylated and controls partially methylated.

For controls unmethylated and 100% methylated, we obtain one PCR band specific and good sequencing results.

For controls partially methylated, we obtain 2 bands PCR (one specific and one unspecific) and very bad sequencing results.

What do you think ?

Thank you

Aurélie

Edited by aurelie, 10 December 2004 - 05:44 AM.


#2 pcrman

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Posted 11 December 2004 - 01:02 PM

Not too bad.

Try raising the annealing temperature a bit to see if that can get rid of the non-specific band.

It is sometimes expected that seqeuncing results are bad especially if there is no much methylation in your sample. Did you get good (clear and strong) band for sequencing? Did you purify it before sequencing? Try using downstream primer for sequencing which may yield better result than upstream primer.




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