My pcr samples are 112bp in size when I am running them in agarose I get bands above 500 bp.
did you design your primers using primer blast or primer ( online programme)
Have you blasted your primers to check that they do not have multiple homology sites all over the genome?
Do you have a positive control that should give the correct size product?
Are you only getting the large band or is it an additional band to the one that you want?
What size standard are you using and can we see a photo of the gel please?
did you design your primers using primer blast or primer ( online programme)
Have you blasted your primers to check that they do not have multiple homology sites all over the genome?
Do you have a positive control that should give the correct size product?
Are you only getting the large band or is it an additional band to the one that you want?
What size standard are you using and can we see a photo of the gel please?
Please use ladder and load them again. 400 bp is very high difference.
@ali
always a pleasure Ali
hopefully we can all manage to sort out a pcr problem
all the best
paul
I have got them ready from other articles, yes I have positive control and I am talking about this positive control. Ali
Are these primers designed for exactly the species that you are working on.? It is still necessary to blast the primers in case of either an error or a misprint in one of the primers from the publication. Do the calculated annealing temperatures agree with the cycling parameters that you are using. It would be useful to see a picture of the sample band and the size standard please. What size standard are you using? Is there any possibility of a mix up between 10bp and 100 bp size standards?
I second to Ali and Paul.
Blast your primer sequence using "NCBI primer blast" to confirm the size of your pcr product being generated by the primer you are using. This will also help to check multiple homology sites which may be the reason for the non specific amplification by your primer.
Dear all,
thank you for your valuable comments, in fact these primers have not been designed by myself. i have got them from too many published papers studied Trichomonas Vaginalis. the sequence of primers are the same but the procedures of PCR are different. I have attached what I have done and could you please give me your suggestions.Ali
hello Ali,
thank you for that information. Your primers are correct for T vaginalis beta tubulin. You are amplifying the correct amplimer. The primers blast to beta tubulin and are about 110 bases apart at their 5'ends so the correct size for the amplimer is about 110 bases
sequence below.
Trichomonas vaginalis G3 beta-tubulin, putative (TVAG_456920) partial mRNA
NCBI Reference Sequence: XM_001582993.1
612
cattgataa cgaagctctt tacgatatct gcttccgtac actcaagctc
661 acaacaccaa catacggcga tcttaaccac cttgtttcc a tggttatgtc cggcacaaca 721 tgc
I cannot think of a good reason for your fragment size of 500 but the primers will anneal at higher temperatures so I might try an annealing gradient up to about 66c and see if the correct band appears but if you have one clean band then although these primers do anneal elsewhere on your genome I think that the most likely situation is that your amplimer is correct but your size standard is the wrong one. Can you borrow someone elses size standard and re run the sample?
regards
Paul
A possibility I forgot to mention Ali. Your protocol suggests that you use 45 cycles in the pcr. This is almost always too many cycles and represents very approximately 10exp12 times as much product as you started with. At these levels the product can anneal with itself to form concatomers of much longer size than the original. If your 500 band is just one of many larger bands or a smear then I suggest that you keep the pcr conditions as you use but rune a pcr where you run 45 cycles and remove a tube every 5 cycles from 30 cycles. It may be that 30 or 35 cycles is a small clean band but more cycles starts elongating the product, Failing this just run a 30 cycle pcr If this idea is right then you may have too much dna in your positive control for the pcr conditions.It may be necessary to run dilutions of the control dna
Thank you very much dear,
I have run my samples on 1% gel and I have done gradient pcr 62-52 and I will my samples on 2% gel on Sunday. Thanks again to both of you Paul and Ali
Thank you dear all, my problem was solved by increase annealing temperature to 60 C.
hello,
i think, as Mr. Paul mentioned, the cause is the exessive cycling which increase the opportunity of non spesific amplification.
thanks
Dear Ali Al-Hisnawi
I send the answer above because I did not see your letter about the problem solving.
Sorry.
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