Hey guys,
I am amplifying an exon in a gene responsible for a hemorrhagic disease
I am doing a PCR with touchdown programm :
95° C 10 min -94°C 30sec,69°C 30sec, 72°C 30sec : 7cycles and then 94°C 30 sec,63°C 30sec , 72°C 30sec and 72 °C 10 min for 29 cycles
after verification on 1% gel agarose i find non-specific bands (i am supposed to find one or 2 bands for heterozygotes but I find 3 or more sometimes) ...
i changed volumes, temeratures and it is still the same maybe i didn’t do the adjustments well it is the first time that happens to me so do you have any suggestion for me please?
Thank you in advance
PS.I don't use seperate Mgcl2 it is included in the buffer and i don't use a hotstart taq i use the classic one.
Note.volumes i use : final 25ul , buffer 2.5ul,dNTP 0.5 ul , primers R and F 1.5ul each , taq 0.1ul and 5ul DNA and water i adjust to 25ul
AND I AM EXPECTING A 595pb product
It is important to know that I took the primers and everything from an article i did not design the primers myself.
Well done Amira Zaier and thank you for letting us know. It always helps to know what works. Good result.
hello
I recommend that you drop the melting temperature every two cycle by 2° starting from 70°C to 60 °C ( 25 cycle )
hope that works for you
You say that your Tm is 60c but in the first paragraph you give the lowest Tm as 63c. What are the Tm values for your 2 primers please as it is hard to say what should work not knowing this. It may simply be that your primers cannot anneal at the annealing temperatures you are using. Do you have a positive control sample that should amplify to use to check the PCR?
Thank you for your answer ! oh yes that was a mistake i am sorry i gave a tm of only one of the primers , as i mentioned before i took all the information from the article , the gene we are amplifying has 30 exons ! I started with one of the exons and i followed all the information mentioned in the article But when it didn’t work i calculated the Tm of the 2 primers to see what can i do with the temperature.
here are my 2 primers :
TGF 26 - ACACCCTGCCACCACCAC= Tm 60
TGR 26 - CTCACCCCAGACACAAGCC= Tm 62
As i know the annealing temperature should be 5°C below the Tm and when we have tow primers we should use the the lowest tm to calculate the annealing temperature (correct me if i am wrong) but i didnt know how to optimize it with the touchdown program when there are two annealing temperatures. And yes i have a positive control and it is always the same result unspecific bands ! sorry for all the details and thank you again so much for giving the time to read all this and to trying to help
Is this a gene in the host genome or are you looking for viral dna in the host please, It makes a big difference as the amounts of viral dna will be much lower and may need a second nested pcr to give good amplification. I would not start with touchdown pcr which is very good for cleaning up a dirty pcr but your problem initially seems to be getting the right band at all so I would do a normal pcr of 30-35 cycles at a low Ta ( maybe 57c) in order to get some amplification of the correct product even if it is a dirty amplification and when you get a band of 595 try raising the annealing temperature using a gradient or then using touchdown pcr or adding DMSO to clean up the amplification. If you still get no amplification with the known positive sample at the lower Ta then make 2 primers just inside your current primers and re amplify your amplified sample (1ul of 1% dilution) with 15 to 20 cycles of nested pcr and even tiny amounts of dna will amplify. Nested pcr is preferable to using more template dna in the first round pcr as dna often contains pcr inhibitors and using too much can stop the reaction. The important thing is to get some amplification then the clean up is much easier but having no amplification there are many reasons for this so I would try the low annealing temperature first. Do you have a picture of your 3 bands...this is not necessarily bad news as heterozygotes do sometimes produce heteroduplex bands which run slower than the homozygote bands and this may be correct if your homozygote is the right size
I can’t thank you enough for your answer ! It seems very helpful i will do as told ! So to answer your question: i extracted my DNA from blood samples from patients with a hemorrhagic disease that i am working on , with salting out methode , i did my dilutions at 20ng and started working with those , i am trying to amplify a gene in the genome and i am not amplifying a viral DNA , i will be looking for genetic mutations as part of my work and will do sequencing afterwards.
Then ignore everything I said about nested pcr as it will not be needed. I would start with amplifying your positive control with a gradient pcr with annealing temperatures of 56 to 64c and see if any of the temperatures give a clean band of the right size or at least amplify one 595 band that can be cleaned up with either dmso or touchdown pcr .
Now everything is clear ! I thought i needed to do it only with touchdown ! I ll try it with the gradient pcr and clean it up with DMSO or touchdown ! thank you so much ! Oh i forgot the picture here it is
That looks like a good amplification Amira Zaier but you need to know which bands are the correct ones so running a size standard (ladder) would make it clearer which are the good bands bearing in mind that if the sample that you are using is a heterozygote for 2 of the bands then the third band ( the top one)may be a heteroduplexof the amplimers. This sometimes happens with strong amplification. Good luck.
To give updates in case it would be useful for anyone else later on that would be in the same situation : so i followed the suggestions below and used lower temperatures according to my Tm that i recalculated (always do the maths even if you take the information ready form the article still some changes are needed for your case) best annealing temperature that worked for me 58 and the lower one 51 (i respected the 7 degrees suggested between both temperatures) according to this touchdown program i stated below i got an amazing clean amplification for all my samples and controls ! Thank you guys so much for your precious advice it worked for me ! and thank you Mr.Paul Rutlan for taking the time to read all that and answer me with all those details
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