Biology Forum › Molecular Biology › Primers Tm
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- October 11, 2012 at 7:18 am #16916
bravebeakerParticipantHello everyone,
I am having some difficulty determining the optimal Tm for my primers for PCR.
I ordered two primers for different genes.
gene A primers Tm 59 and 71
gene B primers Tm 64 and 71
I am trying to run them both at the same time since they amplify almost the same size of a product which is around 240 bp.
I am using KOD-Plus kit.
cycling conditions are 94/2 min
94/15 sec
55/ 30 sec
68/ 15 sec
I got faint bands but at the same time it was smeared, I am using the optimal reaction volume from the kit and I tried it before with other genes so I dont think its MgSO4 or DNA template conc., could it be the cycling conditions? Any help would be greatly appreciated.P.S. I tried to change the annealing temp to 60 and 64 but no use. Currently I am running PCR with 57 for annealing temperature. Will tell you what happens then.
P.S. primers length are (21,23) and (26,23) I want to use them for RT-qPCR after that.
- October 12, 2012 at 1:14 am #112679CatParticipant
Your primer melting temps for each set are just too far apart. Just consider that at say annealing temp of 59, only 50% of one primer is bound to template and ALL (or close to 100%) of the second primer is bound non-specifically (you can estimate the "degree" of non-specificity by truncating that primer to length at which its Tm becomes 59 as well).
If you MUST run with these primer sets, I would try something like adding 2x of primer 1 (low Tm):1x of primer 2 (high Tm) and running increasing annealing temperature range. Go from 59 to 68 in say 0.5 degree increase per cycle.
Let me know what you get as we seem to be running similar assays.
- October 12, 2012 at 5:55 am #112680bravebeakerParticipant
Thanks for the reply will do and let you know what happens.
- October 12, 2012 at 9:47 am #112683bravebeakerParticipant
can I ask a quick question? I checked the PCR machine I am using now and I can modify the run by adding an increment of .5 degree to each cycle starting from 59 C. but how can I assign a range as you recommended from 59 to 68? is it by using the Rotor Q? because I couldnt find the right option in the software. or by using a gradient block PCR? or by simply trying all degrees in seperate runs?
I tried changing the concentrations of the primers as you suggested along with 59 C but still no use for one gene and the other gene is very faint.
Thanks!
- October 12, 2012 at 10:57 pm #112686CatParticipant
It’s something weird – you have to set it to something like "in increment of 0.5 degree for X cycles". 59 to 68 degrees will be 35 cycles in 0.5 increments. Maybe you can try 1 degree increments for 10 cycles and the rest of the cycles at 68.
- October 15, 2012 at 10:46 am #112704bravebeakerParticipant
Today I used gradient PCR and I set the annealing temp to start from 55 and increase 15 degrees gradually. the range was as follows:
55.1 55.5 56.4 57.6 59.2 61.1 63.8 65.7 67.4 68.8 69.9 70.3I also tried 1:1 ratio and 2:1 (Low Tm primer conc : High Tm primer conc)
Both genes were amplified starting from 55.1 until 59.2 and 65.7 after both temps no bands occured on the 2% gel.
Most important observation for me was changing the template DNA sample using different cDNAs acquired from two different treatments in the study.
So now I am thinking of using genomic DNA as a template instead of cDNA. and how about increasing cycle number? any ideas how to get sharper bands?
- October 17, 2012 at 12:12 am #112713CatParticipant
The best way would be to get new primer. All you have to do is shorten the primer with 71 degree Tm until it’s around 60. You said itโs 23b, so you have 8 bases to play with.
- October 17, 2012 at 3:56 am #112716bravebeakerParticipant
Thank you for your advice I will give the current primers a try and will start editing them. I dont want my sensei to yell at me! ๐ Thanks alot!
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