From 1-18-15.
Ran PCR amplicons on the 1% gel (100V for 40 minutes). However, it was a blank gel with only the Hi-Lo DNA marker showing up. None of my amplicons were present on the gel. There are a number of different possible reasons for this.
I only loaded 5 uL of amplicon DNA in the gel and it could be that that volume just wasn't enough to be visualized on a gel. I also did not mix the amplicons with EZ vision loading dye as I was certain that the 2x PCR supermix that was used already contained loading dye. I could try to add the EZ vision dye to a larger volume of amplicon DNA and re-run the gel to determine if either of these factors were responsible for my blank gel.
Additionally, it could simply be that the PCR program that I created in the thermocycler based on what I found through literature review using the same primers was entered incorrectly, or that I mistakenly ordered the incorrect primers (or made a mistake with the primer sequence). This would possibly cause amplification to not occur, resulting in no amplicon bands on a gel. I will double check the primer sequence as well as the thermocycler program just to be sure that those factors were not responsible for my blank gel.
It could be that I made a mistake with the initial genomic DNA isolation. Perhaps I accidentally skipped a step, or used a wrong reagent. I will attempt to quantify the DNA present in my gDNA stocks, something that I should have done initially any ways. I could try to use this gDNA for a different PCR amplification using different primers of known success just to determine if the DNA is viable for amplification. I did originally use a crude genomic lysate of each of these isolates and the AFB primers (16s of P. larvae) successfully before.
//EWW
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