I had this gel electrophoresis result and I was expecting 480bp amplicon size in lane (2). However, I got a band size of 900bp in (2). Taking into consideration that I did not get a band for the GAPDH in lane (1). Lane (3) is a negative control (water). How can I explain the band in lane 2 ??
lovely gel Ahmad. Have these primers worked at these pcr conditions before and does gapdh usually show a primer dimer?
If it usually works then maybe you have mixed up the primers so lane 2 has gapdhf and your reverse primer and does not work but lane 3 has gapdhR and your forward primer which is working but obviously producing the wrong product
Actually I did 2 pcr with 2 different annealing temperature. In the first one I used 50C and I got non specific bands in gapdh lane. While I got no band for the sequence that I targeted. So I changed annealing temp. to 52C and I got this result. Here is attached the first pcr gel
Keeping in mind that the annealing temp. of primers that provided by company is 55C (for targeted sequence)
I am using these pcr conditions for the first time as indicated in literature.
Just to make it clear,, I am using GAPDH as a control,, However my concern is the band in lane (2) it should be 480bp but I got 900bp. Please have a look at image no. 1
Ahmed, it depends on your template. If you amplifying a from a plasmid say pGEM-T (with gene already sub-cloned) and you are using the T7 promoter primers and not your gene primer, then the region within the MCS shall be included. Thus, increase your size of the gene.
But if you used your gene primers and sure, then the following could be responsible:
- the % Agarose you use. Anything above 1% shall make ur DNA run slower
- the molecular weight markers aren't running at the expected sizes
A. Salman
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