I am trying to check the homozygous mutant for Arabidopsis (seeds ordered from ABRC). I have done genomic DNA extraction, using Edward's method and checked the it in agarose, the genomic DNA is there. And also I got quite a good concentration of about 1ug/ul. I designed primers using SIGnal primers design against the salk ids, but my PCR is not working. I have tried different temperatures and polymerases.i have kept the 1st denaturation time about 10 min, and checked the primers, its interacting fine with the genomic DNA using clustal omega. where am I doing wrong?
Try considering the following suggestions:
1. Consider template concentration not more than 50 to 80 ng for a 20 ul reaction.
2. During the initial denaturation steps, add the polymerase after 2 to 3 min. A hot start PCR works well in the case of a gDNA template.
Hope that helps. All the best.
In addition to Sautan ßhow notes, it is recommended to use a gradients of annealing temp as the first step of PCR running, therafyer do a gradient of MgCl2 concentration if you use a master mix with low mgcl2 concentration.
Moreover, you should avoid multiple freeze-thaw cycles of your genomic DNA.
First, check if you can amplify any gene from your samples. Try actin or something that you know always works & you have a positive control.
Salk lines are notorious for NOT having the insert in the correct location (or not having them at all). The seeds are batch-tested & there are issues with cross-contamination of seeds from one SALK line getting mixed into others.
My advice: order ALL of the SALK lines that are supposed to have inserts in your gene-of-interest. Design primers that are at least 1000 bp away from the supposed insertion site. Try using "left border" primers & "right border" primers for the T-DNA.
And be aware that about 20% of the SALK T-DNA lines have chromosomal translocations associated with the T-DNA insertion site.
https://pubmed.ncbi.nlm.nih.gov/21143679/
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