I am getting two bands below 100 bp in addition to the gapdh amplicon . What might be the reason for these bands?
Lane 2- positive control
lane 3-6 - samples
lane 8- negative control
I recommend to run a gel with your primers, just like your negative control but without amplification.In case it differs from this picture, your bands could be primer dimer. You may get rid of them by increasing the specificity (hot start/ higher annealing temperature).
The lower two bands are very likely primer dimers (since you get the same bands in your negative control).
What puzzles me is the second band on top in your positive samples. What's this? Is is an unspecific product or are all the samples heterozygous just in the amplified region (unlikely).
I am so sure it is primer dimer, coz i had that trouble too, and i am agree about running a negative without primers, because maybe you are having contamination in your process or the pcr mix, and you can control it. Greets.
Firstly: You have bands in your Negative control (primer dimers) you must get rid of it by decrease the amount of primer that you added to the reaction if you also got that two bands then increase annealing tempreture or increase the amount of MgCl2.
second: You didn't have constant level of GAPDH expression level try to use another housekeeping genes.
Hi,
And yes, I also agree with the comments. Its the primer dimers and non-specific band is also seen. Try increasing the annealing temperature/MgCl2 or Decrease the primer concentration.
Also, its always recommended in having a PCR control in all cases so that figuring out the issues will be easy.
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