Hi, do you have information about the gene if it's a highly conserved gene? You blasted your sequence of your known gene?

You may in fact not need degenerate primers if the gene is highly conserved or you have good consensus among species.

HYDEN (http://acgt.cs.tau.ac.il/hyden/) is program that allows to design highly degenerate primers. You could check out whether it is a suitable for your application.

You could also give CODEHOP ( http://bioinformatics.weizmann.ac.il/blocks/codehop.html ) a try. This tool worked quite well for our primers.

The gene is for an enzyme, and yes I did run BLAST for it and procured the sequence from another plant of the same family, designed the primers through primer blast but they are not working efficiently, if I perform the PCR at around 50 degree, there is so amplification but when I reduce the temperature to 40 degree, it gets amplified but only 800bp against the expected 1600 bp.

Hi, how are the melting temperatures from your primers?

56-58 degree celsius

Okay, normally you should get a product with an annealing temperature at 53-56 degree celsius. How long are your steps? How do your PCR protocol look like?

My pcr cycle:

94 degrees 4mins, 94 degrees 1min, 40 degree 1min, 72 degree 2 mins, 72 degrees 10 mins, for 45 cycles.

Hi Himanshi, what you can do is when you get this unspecific shorter product:

Increase annealing temperature

Increase annealing time

Increase extension time

Increase extension temperature to 74-78º C

Decrease KCl (buffer) concentration to 0.7-0.8x, but keep MgCl2 concentration at 1.5-2mM

Increase MgCl2 concentration up to 3-4.5 mM but keep dNTP concentration constant

Take less primer

Take less DNA template

Take less Taq polymerase

I hope this can help you!

Cheers, Nadine

Himanshi, I am not too familiar with the plant systems, but consider the following:

is it possible that you are picking up a variant form of this gene? Could there be deletions in the gene in your plant system? Do you know that this gene is expressed in your plant system?

@Nadine A. Gund...thanks a lot mam I will try all these conditions for sure, but the problem is that when I play with the conditions, i don't get the proper amplification.

@Nadine A. Gund.. Infact no amplification at all when I raise the temperature

@Saraswati Lenka... I can say that this gene is expressing in my plant because, it is a gene for an enzyme which is involved in the biosynthetic pathway of production of Isoflavonoids. I just want to fish out this gene from my plant and test it homology with the model system. I think I have to troubleshoot at the primer designing step itself.

Hi Himanshi, but 40 degrees is a very low annealing temperature for your primers. I think you are getting unspecific products... Or (like you mentioned) you design new primers... All the best, Nadine

Himanshi, did you try using any commercial PCR mastermixes? Qiagen has good mastermixes that work with many different primer sets.

Also, I came across the following link (elsewhere on reserachgate), for a software to design degenerate primers:

http://acgt.cs.tau.ac.il/hyden/

It is a free software for academic use. I have not tried it myself but may be it is worth a try.

HI Himanshi

I have few suggestions for you...

1) While designing primers you have used a sequences from some other plant from the same family...you could have used some more sequences to see if the regions where you have designed the primers are really conserved or not....or ceratin positions could have been degenerate..

2) How are you trying to get this gene amplified...from the genomic DNA or cDNA....if cDNA...are you doing OligodT based Reverse transcription or gene specific..go for the second option..

3) If cDNA ....then its quality will be decided by the quality of RNA......by some reason if your RNA quality or cDNA synthesis was not good...you may not end up with complete synthesis of a 1600 base molecule...then subseqeunt PCR may not detect that gene

4) The RNA/cDNA quality issue can also be addressed by desiging two additional primers in the middel of the gene and use them in combination with either 3'-end primers or 5'-end primer.....the fragment towards 3'-end should amplify easily compared to 5'end fragment.....

5) In our lab a well characterized gene we are not able to amplify due to high GC rcontent related issue (check such thing in your sequence)

6) Lowering Tm to as low as 40 may not be good and may amplify other molecules like the 800 bp product.....but as suggested above...you should consider the possibility of a variant also (may be due to Alternative splicing)....you cab still check the 800 bp band..initially by few restriction enzymes...and if needed by sequencing...

7) Finally if 1600 bp is not possible to amplify at a strech ...you can amplify two overlapping fragments and mix them and then try to do a full length PCR

I hope some of these points may be useful to you