i have made a gel electrophoresis for a 7 samples after double arms pcr . the guide paper mentioned that the amplicon is 117 bp but i have seen more than one band in each well
I agree with the previous post. you are replicating non specific artifacts. It sounds like you might need to truly optimize your annealing and extension temperatures. Where did you get what temperatures and total cycle numbers to use? If it was a random guess, that could be the problem. You could also try to use less primer.
From the above image (Supplied) it is seem that,you have primer dimer formation(Due to using higher concentration of primer). Smear bands also appear in the gel photo(its happens when template DNA contain higher concentration or may be incorporated with impurities like protein). Here,As its is Double ARMS-PCR so it will give rise a single PCR product for single sample normally,as primer are mutation specific.So,if two PCR product is seen it may be contaminated by other sample.
In case of primer dilution i normally do this,like After adding recommended (amount mentioned in raw sheet with primer) Buffer or RNAse free water to the primer powder(supplied by company) & then dilutes it to 10 times,like take 10micro litter of freshly prepared primer + Add 90micro litter of diluent(like your buffer(TAE,TBE) or Rnase free water.
Hope for the best.
As others already pointed out, I would also suspect template/primer overloading and suboptimal annealing conditions.
You may want to lower the gDNA per sample, primers per sample, and Mg2+ per sample, as well as increase the annealing temp. In my experience, one of the best ways to estimate the Tm is by using this: http://eu.idtdna.com/calc/analyzer . It lets you approximate the Tm based on your primer conc., ddNTPs conc, and so on.
The gel image is not clear. Check the primers whether they are binding to any other portions of the gene.
all the best
Many people focus in the answer on primer dimers. Many other products may arise; e.g. elongated primerdimers up to 2-, 4 and more fold. GC- rich regions in amplicons may hybridise and become elongated. Mispriming might occur on your template when low copies of targets are present (that's a primer problem). We find many artifacts, i.e. additonal bands, tht occur from renaturating pieces of the target, which become elongated. That was confirmed by sequncing (thats'a target-problem). So you must titrate your PCR very carefully. If you have qPCR facilities, control amplification with intercalating dyes and melting curves!
Probably some other DNA template would amplified along with your gene of interest. Pls check for DNA template contamination in your sample. For example if have plant DNA check with ITS primer any fungal DNA present along with your sample.
1. A lot of smear and a lot of fragments in some cases. Some even show DNA in the wells. A good PCR results should show a single sharp 117-bp band.
2. Probably the quality of isolated DNA is not good enough. Check OD260/OD280 ratio for purity, and check the quality of the DNA (intact or degraded) by running a gel.
3. Use less DNA template. How much DNA did you use for each PCR reaction?
4. Re-check your Tm (melting temperature) and Ta (annealing temperature) setting. The ideal Ta should be a couple of degrees less than Tm. Besides, you should design two primers with 'similar' Tm.
5. Because you are using a paper as a guide (for your primers etc), you need to know whether you are handling the same species or same genome background, and whether you can get the same 117-bp amplicons. With different genome background, you might amplify a different size of PCR product.
check specific primer sequence with sample sequence only one band in the gel
from the picture, its looks like a high template concentration and temperature problem, may be readjustment of annealing temperature and increasing the denaturation temperature\time solve the problem, i suggest using gradient PCR and decrease the template concentration after checking the template purity of course....good luck
Sorry, I can't see your picture. But I think other bands larher or smaller than the expect weight are non-specific band. It doesn't matter if you run RFLP markers. You can increase the temperature to make less non-specific bands.
Try to play around with the annealing temperature. Alternatively, you can purify the expected band from the gel and try to sequence it.
Please check the annealing temp. try with negative control to see If there is any contamination.
Hi people! Just author of question has no attention for our answers! But we know all about this problem. We were learned to check DNA concentration before RCR, we were learned to check primers specificity
and annealing temperature and pH and Mg++ concentration. Happy New Year!
Primer specificity, annealing temperature and final extension are crucial.
The multiple bands you see could be due to unspecific bands or contamination. You should try to check and optimize the annealing temperature and as well set up a negative control.
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