after running RT-PCR, i get the right size of amplified gene fragment, which is a reference gene, but i get also another light bands much smaller than mein, any explanation please??
amplicon size: 223pb
Hi Younes
Unfortunately I could not download the gel but please verify that it is not an artifact, when you are very sure You may cogitate.
If I were in your situation, I will sequence that gene if I had the means if not I will record it very well because from the phylogenetics/ evolutionary biology standpoint, sometimes there is a nascent speciation going on that does not yet express at the phenotypic level but will take time to express, so we can follow the trend.
I don't know where your sample came from but if the gene expressed in a different habitat, there could be a variation eg: mountain mosquitoes from Fokoue, West Cameroon, Africa have adipose tissues beneath their exoskeleton to withstand the cold weather an DNA elution often retain some unexplained bands.
Please investigate these bands further if it is possible so we are reassured. Best of luck
Aline
u should check wht regions do a part of ur primers bind. u'd know wh those are.. for getting right amolicon i would suggest u too increase annealing temperature. it is also possible tht due to less elongation tym it is ur speccific gene only but it is unable to elongate. u should also try increasing elongatio ntym.
my ladder size, is from 50 pb to 1000pb,
Dear Godfred,
i hope that you give some more explanations, about non specific bands and primer dimers??? what do you xant to say exactely???
Dear all,
just some clarifications:
my desired genes or amplified amplicon is there ( the very clear and sharp bands with the desired size which is 233pb)
but olus these bands i got ' you can see it in the photo other bands, 50pb, which are much more smaller than mein, i mean these bands what could be??? dimers??
In my opinion, i guess that there are not dimers, but they are elongation beginning of my amplified genes which could be at this time influenced by some treatments that i did.
Dear Artur burzynski,
Your opinion is very interesting to me
With all the respect to all contibuters,
Best regards to all,
Younes,
Probably primer dimers. You can increase the annealing temperature in order to avoid these bands.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques