Hi all,
I extracted plant genomic DNA from fresh leaves using the CTAB method. Leaves were milled by grinding equipment. The picture shows running extracted DNA on the agarose gel. Picture 2 displays PCR for cas9, and picture 3 is PCR for my desired gene that I could not get any amplification for it. Previous PCR was successful for the primers, so there is no problem with primers. I am running out of ideas. What could cause this issue and any suggestions to address it?
Thank you so much in advance for your help!
the dna is partially degraded but the fragments amplify well as proved by your working pcr. I do not see a no template control (NTC) where you amplify water instead of dna. It should be negative of course but when we see a smear of high molecular weight amplification then it looks like contamination of the reaction with pcr product. Run a NTC sample and if this produces a smear of amplified material then you will need to get rid of the contamination but first run a NTC as well as a sample then take it from there
Thank you, Paul. I marked negative control, with no band in this reaction. Based on this gel, DNA has degraded? Am I right?
Thanks Kyle for your suggestion I will do that. However, I did PCR withe the same master mix and primers that I got bands before. The newest picture is DNA from wild type lines, positive control.
I am finding this gel slightly confusing Reyhaneh Ebrahimi Khaksefidi . Is the small band in some tracks the right size for an amplimer? The NTCs are a real worry. They should be clean but there is a smear of amplified material showing clearly at the top of the gel. It is not completely clear to me if the low molecular weight smears are rNA and you are using too much impure dna or are early signs of pcr contamination. It would be interesting to see a gel with one of your unamplified dna samples ( the same amount as in your pcr reaction).
Overall I think that you have pcr contamination in which case there are some things that you should try to clean up your pcrs.
I would put away or throw all reagents and borrow another researchers taq and all pcr reagents. You will have to order new primers but use new dilutions of the primers made up with another researchers TE or water and using their pipettes and tips not yours. Set up a pcr with 3 no template controls and one positive control or sample. Prepare the pcr using another researchers pipettes,tubes and working area.Use nothing of your own except primers. Also use their pcr machine. While you are running this pcr thoroughly clean your working area and strip down and clean your pipettes. Get new plasticware and clean your pcr machine.. The only sure way to get rid of contamination is to design one primer outside of your primer set but meanwhile try to get a clean NTC and a working PCR but it may take time.
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