I am new to this forum and also new to PCR, I am a student at uni studying molecular parasitology.
I have only done PCR once before and I was handed a working protocol and genomic DNA and all reagents. No thought required!
This time however I have been flung in the deep end with my project and I have very little help.
I am trying to amplify the BoLA DRB3 gene. I have extracted DNA from lung tissue and when I spec it the conc. is approx 2000ng/ul for all of my samples (200).
I made the following master mix for my PCR (based on previous PCRs)
2ul 10x buffer
0.5ul each primer (100ng/ul)
2ul dNTPs (10mM)
1ul MgCl2 (25mM)
1ul DNA (used varying concentrations after realising that the 1st time my conc was probably too high, used 2000ng/ul
then I tried again with a 1:50,1:100 and 1:200 dilution)
Total volume was 20ul (19ul master mix and 1ul DNA)
PCR program was as follows:
94 2 mins, 94 30 sec-57 30 sec-72 1 min (30 cycles), 72 3 mins.
the TM of my primers are: forward 63.3 and reverse 62.
Size of fragment expected 281bp
Any help gratefully recieved! Sorry if its too long but have read other problems and people always seem to ask for more info!