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[epigenetics]

Hello, i am trying to do MSP on Map4 in human. and using Meth primer to generate primers. Map4 has a CpG island of 661 bp. But primers are not working. The main problem i am facing is, if the Tm of primers according to Meth primer are 56/57 degree but IDT showed it 45/ 51. and it is like 7-8 degree differences b/w F and R primers for all of my genes. First i tried to keep annealing tem as of IDT, did not work. Then as of Meth primer, did not work. Tried Nested PCR also. Here i only got sharp band of primers in U and M sample.
I need to give atleast Map4 result to my boss with in 7 days. Please help me out.

Any suggestion how to match up Tm of primers will be appreciated.
Thanks.

[et2b]

epigenetics, on Feb 16 2009, 04:51 PM, said:

Hello, i am trying to do MSP on Map4 in human. and using Meth primer to generate primers. Map4 has a CpG island of 661 bp. But primers are not working. The main problem i am facing is, if the Tm of primers according to Meth primer are 56/57 degree but IDT showed it 45/ 51. and it is like 7-8 degree differences b/w F and R primers for all of my genes. First i tried to keep annealing tem as of IDT, did not work. Then as of Meth primer, did not work. Tried Nested PCR also. Here i only got sharp band of primers in U and M sample.
I need to give atleast Map4 result to my boss with in 7 days. Please help me out.

Any suggestion how to match up Tm of primers will be appreciated.
Thanks.

Hi, with BS-PCRs I dont look at the Tm predictions anymore. To my experience they are quite unreliable. Probably has to do with the fact that the Primer 3 algorithm (that is the one they use) is not entirely exact for bisulfite converted DNA. If it works reliably and without additional bands that is the temperature I use...
Since you can get a PCR product in the area maybe switch from MSP to BSP? Just as easy.
Lot of success!