I have two arabidopsis mutants of SALK and Wisconsin Lox. I genotyped them with LP-RP primers and the LBB and P745 primers to check the homozygous lines. When I've isolated the RNA from it and used the cDNA to run a PCR with the same LP RP primers, I'm getting an amplification of two bands in wild type columbia and a single band in all the TDNA mutants. Can anyone help me figuring out the results?
Dear Bipasha
Some body researching Arabidopsis may be able to help you.
As you have not given the primer sequences and accession IDs. It is difficult to help.
1. Eucaryotes have different forms of same genes due to different combinations of exons, called transcript variants. This may be causing two products.
2. The pcr products may be due to genomic and RNA copies. You can run PCR in the RNAs without reverse transcription to confirm if the gDNA contamination in your RNA.
Thanks.
Did you BLAST your primers to make sure they are specific to one locus? Arabidopsis is technically diploid, but had a rather recent genome duplication so it can be hard to design primers that are specific to one locus. My bet is that you have a closely-related pair of genes that both amplify with your forward & reverse primers.
The LBB primer is a bit prone to PCR artifacts, that's probably why it shows up in your WT. Increasing the annealing temp can help.
Why are you doing PCR on cDNA with T-DNA primers? That's an unusual approach, what are you hoping to find out?
If you really want to determine what you are amplifying you could sequence the PCR products.
Amy Klocko It was actually an experimental mistake that we had made. I was supposed to use one of the primers LPor RP and design another primer 150bp away from the LP/RP sequence for qRT.
Quick note: make sure your cDNA has been treated with DNase or you can get all sorts of gDNA artifacts coming through during qPCR.
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