Hi to all
Hope you are doing well.
I am amplifying Kan cassette in PkD4 plasmid with 40bp extensions in primers to knockout gene in MG1655 E.COLI by lambda red mutagenesis . the expected product size is 1540bp. As the primers are long so Tm is around 72c for both primers. I run PCR two times by setting annealing temperature 67c and 60 respectively. But both times I get very faint band for product but brighter band for primer dimmer.
On left side I loaded whole 50ul of PCR volume but still very faint product. On right side I loaded 5ul and get very very faint band even you can't see.
Can you please tell me the reason?
I am using Dreamtaq green polymerase,
1% gel, 30 cycles PCR. 5 ul of plasmid with concentration 60ng/ul.
Run the 1kb plus leader on both sides
Try a 2-step PCR combining annealing and extension into one step at 72°C for 1 min, and use less template 300 ng is an awful lot for a plasmid, 10 to 100 ng or even less should be enough. If the sequence is very GC-rich try adding 3-5% DMSO to the reaction.
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