Hi, you are sure that your primer should amplify a product with a size of 700 bp? The only thing you can do is to cut out the band and sequence it. Blast it and then you will know what you are amplifying. Cheers, Nadine
Yes but otherwise you won't know what you are amplifing!! Or you design new primers!!
Ok, I can try sequencing, thanks Nadine,
what about the noisy background in the same lane, do you have an idea to get rid of that ?
Yup, give me some time to dig it out from my Laptop, I will upload it,
I hope you have tried gradient pcr to optimize the temperature. May be improper annealing temperatures and extension time is causing this problem. Also.. did you check if there is an intronic region spanning between your primers. So may be when you designed it you designed considering it and now when you amplify your cDNA.. that is not there.. ? or there might be problem with your RNA integrity.. and hence you get short cDNA fragments.. Have you chekced that ?
Sorry, Nadine the pic on my laptop is a bad and faint one, since it does not meet my interest so I did not care to capture good photos for it but I can search for a good one in imaging device CPU,
Shrey, I tried different annealing temperatures, started with 50 / one minute and it was not good, and changed to 55, also non specific, today I tried 58 hopefully I can find better result tomorrow,
For RNA quality, it was total RNA not vRNA, extracted from whole blood (Qiagen), I did nanodrop quantification, 147 ng/ul, 260/280 was 1.85, so I think it is not bad,
I am using extension time 72/2 minutes for 55 cycles ( according to certain paper I follow), final extension 70/10 minutes,
Also, I tried seminested PCR, also not good, not the expected size,
For primers, they are the same as stated in certain paper, so no chances for bad primers,
What is the relation between extension time and my case ? can you illustrate this please ? I never think about extension time, usually target the annealing temperature,
The rule of thumb for PCR extension time is 1 min per 1 kb and I believe that what you have been using for your PCR protocol is perfectly fine but for products
Your protocol sounds fine. 2 minutes extension is a bit long but even in that case it should not give you 500 bp fragment. Usually for 700 bp, 45-60 sec as said above is sufficient. Greater extension times gives unspecific products (longer) along with your specific ones.
and are you sure that you designed your primers using mRNA sequence and not genomic DNA sequence ?? It might be given wrong in the paper. Check it once to rule that out. Also check for splice variants. Is your primer lying in a region of potential splice variant? Also, the nanodrop values of RNA look fine but run a gel to be sure. Nanodrop may give wrong results sometimes. Have your tried using a different RNA or a positive control for your gene ?
I think the problem may be in the primers ?!!, but how that paper write bad primers ?
BTW, I did TOPO cloning of this band and I did obtain the band at specific site after plasmid digestion, then followed Nadin opinion to sequence, but unfortunately it was junk or cellular genes.
I should follow my supervisor advice from the first to through these bands in the garbage, because they are non specific and may be junk or cellular genes !
Did anybody before face the possibility of bad primers wriiten in a paper published in good journal ?
Nadine, please take a look on the bands after cloning, but by sequencing they were fake !!!
Yes, many times.. So mostly I try to design my own primers. You can always do a primer blast for your desired genes and the primers you picked up from the publication to see their quality and then if they look fine to you.. go ahead..
Can anybody give me an idea about gradient PCR and its protocol and how to adjust using thermal cycler ?! thanks
HI Ibrahim, do you mean the fat upper band? Or the lighter lower band. Cheers, Nadine
the upper band is for plasmid T/A vector, it is not the case, I mean the lower band, 4 bands at 700 bp but there is other bands at 500 bp (these are non specific), my expected site is 670 bp,
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