I need help to determine the Tm of this primer pair Primer F 5' ATCCCAAGCCTTCTACC 3' Primer R 5' GCCACAGCAGTGTTTTCT' 3' I tried several annealing temp. 47 , 50, 51 and 52C but no results.
Is there anyone who can calculate the exact Tm suitable to these primers? I contacted the company but no response.
I always used "Nearest Neighbor" calculation as it is for example calculated by this program: http://www.metabion.com/support/biocalc.php. This one gives 46°C and 47°C for your primers. Because you already tried 47°C your problem may be elsewhere. Primers work best if they end on a C or G with an A or T next-to-last. So, your first one has a very "strong-binding" end and the other one a quite "loose" end. Maybe you can adjust this. What about your DNA, is it genomic? If so, you may have to prolong the first denaturation step to give better accessibility to the region you'd like to amplify. Which enzyme do you use? Taq in general gives higher yield (but includes errors). If it is like a Pfu polymerase you may add additives like Betaine (almost always enhances output) or some DMSO. Would you mind adding some details on your PCR? We may give you more related hints then. Best wishes!
Hope this helps, I use it to calculate Tm.
http://www6.appliedbiosystems.com/support/techtools/calc/
Or you can use this simple formula to get a general idea:
Tm = 4°C x (number of G’s and C’s in the primer) + 2°C x (number of A’s and T’s in the primer)
I go with the formula suggested my Mr.Jon.! He is correct! I apply same formula too. have You tried gradient PCR... ? if not try it...Best wishes!
I always used "Nearest Neighbor" calculation as it is for example calculated by this program: http://www.metabion.com/support/biocalc.php. This one gives 46°C and 47°C for your primers. Because you already tried 47°C your problem may be elsewhere. Primers work best if they end on a C or G with an A or T next-to-last. So, your first one has a very "strong-binding" end and the other one a quite "loose" end. Maybe you can adjust this. What about your DNA, is it genomic? If so, you may have to prolong the first denaturation step to give better accessibility to the region you'd like to amplify. Which enzyme do you use? Taq in general gives higher yield (but includes errors). If it is like a Pfu polymerase you may add additives like Betaine (almost always enhances output) or some DMSO. Would you mind adding some details on your PCR? We may give you more related hints then. Best wishes!
I don't know what your target gene or region is, but I tried to find it back using UCSC in-Silico PCR (http://genome.ucsc.edu/cgi-bin/hgPcr). Unfortunatelly, there was no specific region complementary to your primers. This means that "no genomic region may be amplified by your primer pair". Probably something went wrong during your primer design. May I syggest that you redesign your primers first. Once it's done, you may then check for SNPs using SNPCheck3 (https://ngrl.manchester.ac.uk/SNPCheckV3/snpcheck.htm) and the specificity of your primers using the UCSC in-Silico PCR. As you know, your primers should be the most specific for your region of interest and should not contain SNPs near the 3' end to ensure a succesfull experiment. Hopefully this will help.
using OLIGOCALCULATOR software it is 47 and 48 for forward and reverse with no self complimentarity. using NCBI BLAST primers have shown significant similarity to newcastle viral strain with high query coverage but higher e value. of course with only 17-18 nt e value must be higher. i think Mr. Sultan is trying to amplify viral DNA.
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