I have problem with my gel i hope to find someone to help me, there is smeared band in with amplification of S16 rNA in whole blood samples. i have put gel picture in attached file , the problem in 17 line,
Hi,
It appears that you are getting some amplification, but not of a defined band. In other words, you have some non-specific amplification, where your primers are extending from the wrong site. The usual advice is to adjust the annealling Tm or time. As well, you could consider
1. changing the primer design
2. changing to a different Taq
Non of your reactions look great, so rather than worry specificically about lane 17, I suggest you work for a generally improvement in the reaction, testing everything from template prep quality to primers and thermo cycle. If you can improve the general reaction, then I think your problem in lane 17 will solve itself.
i agree with Liam,
your pcr has a problem with primer dimer so the primers are getting used up annealing to each other and not amplifying your sequence. You can try many things including less primer, quicker set up of your pcr or probably best to use a hot start enzyme or add your enzyme while holding the reaction mix at 80c. Possibly also designing better primers which do not self anneal but when your pcr is working properly you will find that poor amplification disappears. First try should be to buy/borrow some hot start polymerase and see what the product looks like. If the problem persists you can try reducing the annealing temperature 1 or 2 degrees in case where you get a smeared product it is because one primer is not annealing properly. If that fails try adding DMSO at final concentration of 5% and run a gradient of annealing temperature s which may clean up the product but first get rid of that primer dimer
Liam and Paul has given you very good suggestions.
It does appear that you have way too much primer added? the template and primer ratio is very critical for amplification. Have you tried optimizing your PCR condition by using varying amounts of template and primer ratio? if you have not, I would set that up in addition to trying varying annealing temperature and added reagents.
Good luck!
But there is amplification in Lane 1,4 and hazy in Lane 9. Setting up a gradient PCR and finding out the annealing temperature where there is proper amplification can be done. Too much template in the reaction will also mess up. Generally 100-150 ng is sufficient to amplify .
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