Aysegul Gungor Aydin It is good to follow all the rules to design a pair of primers. However, in my experiences, some primers can still work perfectly even they are not perfect (not designed according to standard rules). Just order 1 or 2 reverse primers and check them out. However, Tm of both primers are important (should stick to its basic rules, such as similar Tm values for both forward and reverse primers).

Hi Aysegul Gungor Aydin, As I can see that if you're aiming to amplify the entire gene, then you're left with not many choices but to design reverse primers for the end sequence of the gene. (You can do nothing if GC Content is high in the gene sequence).

So, do not stress much to maintain the GC-content, rather focus on the Tm and the probability of formation of 2o Structure and Primer dimer.

There are few free tools to check that -

1. IDT-Oligo Analyser Tool - https://www.idtdna.com/pages/tools/oligoanalyzer

2. NEB Tm calculator - https://tmcalculator.neb.com/#!/main

Best wishes.

I agree with Yuan-Yeu Yau about ordering 2 primers. Firstly as he says the rules for primer design are just guides and many other primers will work. Preferably with annealing temperatures similar to each other. Also if you amplify with the outer reverse primer first it allows you to re amplify the possibly dirty product with hemi nested pcr with the inside of the 2 primers.. If you still get poor amplification there is still touchdown pcr,dmso and gradient annealing to improve the results and primers for pcr are cheap.

First use primer3plus software for design both forward and reverse if you could not get a good result for each primer at your desire place, now you could breakdown the rules of primers and order a primer for reverse wich is not same in temperature with forward, even to 4-5 degree differences, now you should change your program for PCR from simple to touchdown. this PCR is really fine and with the range of decrement or increment you set for it could use both primer out of their real temerature. I always give sharp band with touchdown PCR.

if you use high quality polymerase such as Q5 and also GC enhancer (if your fragment is GC reach) you could reach your desire sharp band even to 6-8 kb without any doubt.

furthermore for large fragment you could use different ways such as overlap PCR with some sets of primer which is hard and dont suggest you.

good luck

Hi,

I advise you to design primer using primer3.

primer3.ut.ee/

Hey,

As far as I understand you want to amplify whole gene to clone it ? And I also think that you will you cDNA to amplify it. Otherwise you cannot amplify a whole genomic sequence by a PCR. No of the enzyme will be able to amplify it.

I can advice you to first gene spesific cDNa synthesis ( basically use your reverse primer in RT reaction in very low concentration such as 0,5 uM). after you synthesize your cDNA you will be eliminate many other non-spesific production which can be created by binding of your forward primer to 3' regions.

Second you can perform the PCR now, with even tm differences due to eliminating many other off-targets.

But. If I understand you wrongly and if you want to amplify whole genomic region. It is impossible since the gene is really huge with introns.

Lastly use an enzyme like Q5 DNA polymerase (NEB) it has realy high capacity to amplify GC rich regions etc.

Thanks for all your great advice. First I am using cDNA to amplify KCNA1 and Platinum™ PCR SuperMix High Fidelity to reduce set-up time. I have already designed the primers following all the rules to design a pair of primers with 1.5-degree differences. To amplify the entire gene I had no choice for left primer, however, I picked the right one in 3'UTR by using SnapGene. Even though I tried different primer concentrations and annealing temperature, I could not be able to amplify it. When I ran my primers through NetPrimer, it indicated some potential issues with the reverse primer. At this point, I have to redesign my reverse primer but can not figure out how to find a good pair. On the other hand, I decided to add the sequence GTTTCTT on the 5' end of reverse primer for efficient TOPO cloning. My next question is that when calculating the Tm of my reverse primer, should I only use the bases which are complementary to my target, or should I also include GTTTCTT extension on the primer? Thanks in advance!

Hey,

Then as I explained use gene spesific primer for cDNA synthesis if you have non-spesifity problem. Since I dont know your RT enzyme I cannot tell anything more in that point.

Second, you cannot use this enzyme to amplify a GC rich region, please read the enzyme properties. I advice you to change it with Q5 (NEB). Also your enzyme's amplification fidelity and accurity are also low. Accuracy (vs. Taq): 6 X

GC-rich PCR performance: Low.

Third, no you dont calcualte GTTTCTT for tm. However if you want after 3 cycle you can increase the Tm that contains GTTTCTT sequence as well.

lastly but not least, You can never find a good primer for your cloning experiments. You just pick the best you can. If you really require high amount of product, once you amplified the product, you can cut it from gel and purify. Then you can use your PCR product as a template to produce efficient high amount amplification.

In my opinion your problem is not primers, your problem is the enzyme. Please always read your enzyme's instructions and specifications. All enzyme contains a specific purpose this is why we have a lot of DNA polymerase variants.

I forget the mention.

If you cannot change your DNA polymerase, go nested PCR.

first amplify by using 5' and 3' primers from outside of your template with low GC ratio and nice Tm compatibility. Then use 1 ul from the PCR reaction as template with a second pair of primers so amplify specific sequences you want.