rt-pcr

[bhunni]

iam getting more than two bands in rt-pcr products. what is the reason? should i have to alter the temperatures?

[danfive]

You're getting non-specific products. Optimize Mg++, Annealing temp, primer/rna concentrations.

[superari77]

There are many possibilities... First of all try to optimize the reaction rendering it more specific (essentially by incrasing the annealing temperature), but is also possible that you are getting another product (such as a splice variant, or, if you are using polyploids, homeologous products, or are you amplifying a gene belonging to a gene family??).

[miss-pcr]

try to check Mg++ conc. and the contamination.

[bhunni]

Originally Posted by danfive

You're getting non-specific products. Optimize Mg++, Annealing temp, primer/rna concentrations.

thank you .I tried. Mg++ optimiztion worked out.

[bhunni]

Originally Posted by superari77

There are many possibilities... First of all try to optimize the reaction rendering it more specific (essentially by incrasing the annealing temperature), but is also possible that you are getting another product (such as a splice variant, or, if you are using polyploids, homeologous products, or are you amplifying a gene belonging to a gene family??).

Thanks a lot for useful suggestions.
I am not amplifying a gene belonging to a gene family.

[bhunni]

Originally Posted by miss-pcr

try to check Mg++ conc. and the contamination.

ur suggestion was useful