Hello every body. I want to set up my experiment for Methylation-sensitive high resolution melting (MS-HRM).
When we used conventional PCR mastermix( YTA- Yekta Tajhiz Azma) I gained my interest specific band, while when we used HRM mastermix (solis biodyne) I had 2 band, which the nonspecific band(around 120 bp and it is not the primer dimer) is sharper than specific band(210 bp). I used different concentration of mgcl2, gradient annealing temperatures But the problem of the presence of nonspecific bond were remains.
How can I fix this problem?
I am grateful for your advice.
Ashkan Faridi different pcr mastermixes have different protocols different polymerases . just follow the protocol correctly with the correct concentration for all ingredients . if you have trouble , look at protocol troubleshooting or contact support emailing the company you bought the enzyme and this avoid you to waste time trying various things that may not work. stick with the pcr master mix that gave you the bands of your interest .
Dear Mr Faridi. This happened to me when I used a second brand of mastermix (which has already expired, but unopened) and a third brand of matermix (not expired, but freeze-thawed multiple times). It's either the Taq polymerase misbehaved (i.e. chaotic) or its activity starts to decrease. Best to aliquot your mastermix into different tubes to reduce the potential of multiple freeze-thawing and use mastermixes which have not expired.
dear Florian C Priller thanks,
we play with annealing temperature around 54-65
thanks dear Nurul Adhwa Rahman.
but we buy the mastemix a few week ago
and we sure that it is not expired or freeze and thawed.
all the best
Dear Faridi,
I would do a simple check to the reagents used in both PCR master mixes used and see what they differ , then used this information and try to optimize as much as possible due to the fact that different enzymes / reagents from different companies can have different needs, however keep in mind that is better to use only one of those PCR mastermixes for your analysis in order for you to have the best reproductability in the future.
Good luck :)
All of them, depend on the your control primer tm , because HRM is so sensitive case
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