I have a problem with my PCR amplicon size. The desired size is 758 bp, while I am getting a band size of 1200 bp. No amplifications in the negative control. But in each case, I am getting a band size of 1200bp. What will be the logic behind this?
what is your dna ladder. It may be that your gel is too high in agarose and the size ladder too small and then sizes at the high molecular weight sizes are quite inaccurate. You need a band in your ladder quite close to the expected amplimer size for an accurate sizing
Paul Rutland I used a 100bp ladder and 2% agarose gel for this.
ok the gel concentration is good and your amplimer should be within the range of 100bp ladder. I am concerned about the smears at the lower part of the gel which could be degraded rna or dna in which case I wonder if you are using too much template dna and your upper band is genomic dna and there is actually no amplification at all. Do you have a positive control sample that works in this pcr reaction. Could you dilute the amount of dna that you are amplifying and run a sample in the gel just to make sure that these larger bands are not dna present in the dna sample that you are amplifying. do you know (in ng ) how much dna you are amplifying and do you have an OD260/280 to check the purity of the dna?
The last two bands are positive controls that I used for this study. nearly 500ng DNA template was used for this and I didn't check the purity of the DNA.
I am using primers for simple PCR amplification from a previously published paper. In a published paper, the author reported the PCR product Size of about 747 bp. Can it be possible gene construct varies from organism to organism so different organisms will show different bands in PCR after using the same primer for a specific gene?
Thank you for your answer.
I think that you are using too much dna which often leads to non specific primer binding but that probably is not the cause of the band size and that some of your dna is actually rna. BLAST the primers and check that the size reported is correct.There have been many errors published in the past and this may be one of these
It seems you have no amplification in this experiment. The bands you are assuming as amplicons are DNA templates. I would recommend optimizing the working DNA concentrations by employing higher dilutions and annealing temperatures, possibly through gradient PCR.
I agree with all troubleshooting guidelines here, I'd also suggest you confirm the primers computationally(i.e Insilico pcr), you will be certain of your amplimer base pairs on your your DNA template (if it got's a sequence genome template). Take note, published primers amplimers size aren't 100% accurate.
I will agree with the above comments, I do not see any amplification in this gel. What was the concentration of template DNA you used?
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