PCR product size - (Mar/30/2011 )
I have a forward primer started from nucleotide no. 79 till 99 and a reverse primer located at nucleotide no. 114 till 391. From there, how can I predict my RT-PCR product size (from cDNA)?
I have designed my primer gDNA sequence where there is an intron within my primer design. Should I exclude or include the intron sequence in order to count my PCR producr size?
From your explanation it seems to me that predicting your RT-PCR product should be pretty straight forward: just count the number of bases from the start of your forward primer to the end of your reserve primer using the cDNA sequence of your gene. You should definitely the intron sequence.
you didn't finish your sentences. Definitely include or exclude intron sequence??
I got the bands for my gel electrophoresis but I didnt know whether they are the right bands or not. Can I have your email so that i can show to you the picture?
ifhmn on Wed Mar 30 13:59:44 2011 said:
I have a forward primer started from nucleotide no. 79 till 99 and a reverse primer located at nucleotide no. 114 till 391. From there, how can I predict my RT-PCR product size (from cDNA)?
I have designed my primer gDNA sequence where there is an intron within my primer design. Should I exclude or include the intron sequence in order to count my PCR producr size?
Is your reverse primer really going from 114 to 391? I mean, no way you have a primer that's 277nt long, do you? I guess that's in the gDNA, so what's the actual location in the cDNA, ie WITHOUT the intron.
To calculate the size of your RT-PCR product you have to substract the start position of your forward primer, to the start position of your reverse. BUT, you need to look at the size in cDNA, by definition cDNA will not have introns, as is the copy of your mRNA.
I'm guessing the intron in between nucleotides 114 - 391, remove that, and calculate the actual size.
Sorry,my mistake. From my cDNA sequence, the forward sequence start at base no. 69 till 89 and reverse sequence start at base no. 395 till no. 414. From the gDNA seq., there is an inton between the primer with 188bp.
ifhmn on Thu Mar 31 10:07:10 2011 said:
Sorry,my mistake. From my cDNA sequence, the forward sequence start at base no. 69 till 89 and reverse sequence start at base no. 395 till no. 414. From the gDNA seq., there is an inton between the primer with 188bp.
Do you mean there's an intron between your forward and your reverse primer?
If so, your expected product from cDNA should be 414-69 = 345bp you add the intron to that 345 + 188 = 533bp which will indicate that you have gDNA contamination.
Fo a primer to "expand and intron" it should bind to the end of one exon and the begining of the next one, wihout binding to the intron. That way, those primers will not be able to amplify from gDNA as the intron wont allow the primer to bind.
hope this helps