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methylation specific PCR

no band

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#1 SF_HK

SF_HK

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Posted 22 August 2021 - 06:34 PM

Hi,

 

I would appreciate any help. Im having trouble with MSP.

 

I have done bisulphite conversion using 1ug genomic DNA. But Im not able to get any bands on my gel following PCR at various conditions:

 

1. 0.5um or 0.25um primers

2. initial denaturation tried at 3 different conditions: 95 deg for 2 min, 5 min and 10 min

3. 35 cycles at following:

-denaturation step at 95 deg for 30 sec

-annealing temp tried at 3 different temp: 55, 60, 63 deg, for 30 sec

-extemsion at 72 deg for 30 sec

4. final extension tried at 5 min and 10 mins

 

The target PCR band is 180bp

 

I did check expression of GAPDH using genomic DNA (prior to and after bisulphite conversion) and I got my target band.

 

A. Im assuming I have to design new primers? The only software that has been able to come up with possible primers for MSP is methprimer. Other websites say no primers found for MSP? am'I doing anything wrong?

 

2. Is there any way I can check if my bisulphite DNA has been converted well enough?

 

Thank you

SF






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