I'm in the step of primer design for cloning gene from genome of yeast Hansenula polymorpha. Because of my desired gene sequence in Genbank that provide only exactly sequence and don't have any upstream or downstream sequence, and I want to clone all provided sequence.
Then, I design primer that flanking with restriction enzyme site, and check Tm, GC, delta G etc. by Oligoanalyzer.
My question is should I put primer sequence with RE site , or only primer sequence to Oligoanalyzer for checking the parameter? because after I put only primer sequence, the parameter is ok, but If I put primer sequence with RE site, many parameter is not good, and I can't move the sequence much because of the exact gene sequence and I want to clone all of gene sequence that provide in Genbank.
Best regards,
Tatpong
RE sequences are mostly palindromic which is why the self dimer and hetero dimer do not look so good. In any case if the primer is specific than it will bind specifically against the gene sequence so if that part looks good and as long as you are able to amplify the complete sequence it should be fine.
RE sequences are mostly palindromic which is why the self dimer and hetero dimer do not look so good. In any case if the primer is specific than it will bind specifically against the gene sequence so if that part looks good and as long as you are able to amplify the complete sequence it should be fine.
I encourage you to add the restriction sites into your sequences to be analysed in your preferred program/application for oligonucleotides design, to allow your program/application to give you the best calculations for your oligonucleotides, since if you do not add the sequence of the restriction sites the parameters such as Tm and secondary structure are not going to be calculated accurately.
Regards
I usually use DNA calculator of Sigma http://www.sigmaaldrich.com/life-science/custom-oligos/custom-dna/learning-center/calculator.html to check my primers. To amplify the fragment for cloning also my primers carries RE site, that increase the secondary structure and the probability of dimer formation. Very often in this case my primers have strong secondary structure and high melting temperature (about 68-72°C) and unfortunately I can't change the sequence if I need whole gene length and only one/two RE sites are ok for my cloning. The solution of this problem is to perform two step PCR. In first 5 cycles of amplification I use the lower temperature Tm (calculated only for the part of primer that binds to my gene), and then other 30 cycles in which the Tm is calculated for entire primer (with RE sites). Usually it works for me even if the secondary structure is strong. Good luck!
Dear Manchiganti, Barraza, and Maciag,
Thank you for your suggestion
Pleasure :) .. do post back here, if it works out for you.
Best,
Rushiraj
Dear all,
Can we keep the primer length upto 30 bp while designing, if we are including RE sites for cloning and GC base pairs upto 2 in flanking region for maintaining GC content? My concern is that by increasing the length of primer (by taking sequences complementary to the target region), it will provide the specificity to the target region but on other side, Tm will increase more and so secondary structures.
Hi Tripti,
According to my view, Yes you can choose a primer for cloning upto 30bp. Because in that u r adding 6 bp restriction sequence + 2/4/6 bp GC content to maintain Tm of primer sequence. So main sequence of your primer is 20 to 22 bp. So i think 30bp sequence will not create any problem.
Good luck !
Hi Tatpong,
Primers with RE site have higher Tm and probable worse secondary structure compare to a shorter primer.
I suggest to do a touchdown PCR. If you can use PCR machine available with this feature. For the annealing temperature just set the highest temp for exmple 65-70 °C. Set the PCR to decrease the annealing temp by 0.5 °C after every cycle down to the calculated annealing temp of your primers. For example 55 °C (lowest temp). Run the PCR on this temp for the following calculated cycle number. For example: (total cycle number)-(numbers of decreasing temp cycles) = (remaining cycle number). E.g.: 40 cycles - 20 cycles = 20 cycles at 55 °C. This technique not as elegant as gradient PCR, but it can be usefull. Unf you won't know the ideal annealing temp like in grad PCR, but it will maximize the amount of the specific product.
Or you can use less efficient blunt end ligation (anyway from my experience with this, it isn't worse than sticky end ligation in my cloning experiments). Con is that the reverse direction built-in's prob is 50 %.
Other hand you can use TA cloning. E.g. Taq DNA Polymerase hasn't got 3'-5' proofreading activity. Therefore it leaves A-overhangs in 3'-5' ends of PCR product. You need to singe cut your vector with a blunt end RE, then put T-overhangs to 3'-5' ends of the linearized vector with terminal transferase and ddTTP. You can use TA cloning kits. Main pro is that simpler than sticky end producing RE and ligation. Maind con is that the reverse direction bulit-in's probability is 50%, like blunt end ligation.
Bests
Tamas
PS: I think you can use pur RE primers with touchown PCR.
so we have to maintain Tm including RE sitting site plus RE cut site plus genomic sequence of primer right?
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