I have 10 rice samples which I'm testing with housekeeping primers.
9 of them are supposed to be transgenic. And 1 is wildtype.
I did a PCR with all of them, in which I had a band with all the samples except the wildtype.
My wildtype sample DNA concentration is around 280ng/ul which is way too much than my other samples. Now I want to perform a PCR again but with diluted genomic DNA of my wild type.
But I don't know exactly which protocol should I be following to dilute my genomic DNA of the wildtype sample. Any help would be appreciated.
Regards,
Swati.
As a first approximation use the same amount of wt dna as you are using in your samples. Failing that try 10,25 and 50 ng of dna and see what works best for you.
Hi Swati Jagani
Here are some steps to confirm DNA quality and dilution for PCR
- Run a gel with equal volumes of total DNA from all tested samples. Make sure you have not much DNA degradation
- Using Nanodrop or OD equipment to measure DNA concentrations. Find if it is consistent to the gel running result.
- Using water or TE buffer to dilute the DNA to concentration from 50-70 ng/microliters
- Run a gel again using diluted DNA (10-15 microliters per well). Find if all samples show the same bright bands
- Run PCR with regular primers that you know it works well with rice DNA
As you have good PCR products from all samples, you can perform experiments with other primers.
Good luck!
- Badges
- Science method