I have used primer for rmpA gene in hvK. pneumoniae. Primer sequence is rmpA F-P 5’-GGCCTAAAGCAGTTAACTGG-3’ and rmpA R-P 5’-CTAAATACTTGGCATGAGCCATC-3’. Recipe used are as follows
Master mix 5 µL
Forward primer 0.2 µL
Reverse primer 0.2 µL
Plasmid DNA 1µL
And annealing temperatures tried were ranges from 51 °C to 56 °C
Yousef M Abu-Zaitoon
You need to work out the concentration of primers and I suggest it to be 10 uM. You should note annealing temp. of primers came from the company.
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Paul Rutland
your reaction volume is too small. evaporation is probably making it even smaller. try using a reaction volume of 20ul or more. That is easier to work with' Do you have a small size diffuse band after amplification?
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