Hello,
I'm trying to improve a PCR reaction. I sometime have one of the sample that get a "perfect" amplification, but most of them are just weak with primers left.
It does not depend on the quality of the sample (for the working sample is random, and if I run again the same reaction, that sample won't work better)
I tried different way of pipetting or mixing, no differences... Has anyone any clue?
There are a few little things to do to improve PCR yield and quality.
It's very simple process to optimize your PCR
-look out for primers to be used, using appropriate software
- optimise annealing temperature by 1-2 degrees centigrade
-chose a satisfactory primer concentration
- increase the cycling number (40-45)
-hot start the machine manually
Pls confirm the procedure using the appropriate guidelines.........cheers
you have weak bands and also you have smear.
did you check your DNA con?
these smear are look like that you ran a total DNA
Alireza: Thanks, I actually look for a smear, it is something close to the first step of AFLP. I tried with higher DNA concentrations, It reduced the amplification…
The short bands are most likely to be conserved area, I'd prefer to have the bigger PCR produces amplified
if increasing dna caused lower amplification this suggests that your dna contains pcr inhibitors. Try running the pcr with slightly more Mg and using less dna ( or repurifying your dna) The od260/280 ratio may indicate the presence of contaminants and should be about 1.8, Also are you running a no template control sample to detect pcr contamination problems
if your quality of your DNA samples is good for having more produce you can just increase final volume of your pcr
Based on the ladder result, I can tell that the electrophoresis conditions were perfect! I think you have to re-purify the extracted DNA, and I recommend column purification kit. If its a plant DNA, this is a common issue!
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running