Dear all,
I am trying to get a homozygous from Sail line stocks. Plants are the third generation from a homozygous line, and I am still unable to detect any clear or correct bands.
It seems that there are primer amplification problems; I repeated the PCR three times and increased the annealing temperature from 58 to 60.
There are many ways to stop a pcr working and 2 of these are impure or excess dna and primers that form a dimer so remove the primer from the reaction. Check that your dna has an od260/280 of close to 1.8 and that you are using between 5 and 50ng dna in a 50ul pcr reaction. Scale down as necessary. For dirty dna samples use less dna which should mean less pcr inhibitor and better results. The small band on your gel is primer dimer so either use a hot start enzyme and/or use less primer...maybe half what you are using now. usean annealing temperature 5c below the calculated melting temperature of the lowest melting primer.
If this fails try adding 1mM more Mg to your reaction to counter the effect of pcr inhibitors.
Always run a positive control and a no template control (water not dna )
OK Maryam Mz above 2.3 is not good. It might be rna in which case you do not know how much dna you are adding but more likely you are using column purifications (CTAB is probably better) and you are getting guanidine salts in the eluted dna which can spoil a pcr. If this is the case then try purifying less crude tissue and do 2 washes to get rid of the salts before eluting the purified dna with elution buffer or water whichever your kit uses
Paul Rutland I am following a quick and dirty genomic DNA extraction. It can easily be used for genotyping with PCR o many samples trying to get a homozygous line for my mutant. I'll still need to puri some before running PCR, and I am thinking of reducing the DNA volume from 1ul to 0.6ul. Today's samples also had the same issue, unfortunately ( od 260/280 range between 1.9-2.2). The protocol I follow is: adding DNA extraction buffer, adding 300ul fo isopropanol, and then washing with 70% ethanol 700ul), all shaking tubes in this step. Thank you so much for your great reply.
Some of the KOD polymerases are particularly good for amplifying dirty dna. Using less dna I would run 4 or 5 dilutions of your dna ,1, 1/2, 1/4, 1/8 and 1/16.
Sometimes with pcr inhibitors less dna means less inhibitor and more product
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