By far the easiest is to use any software made exactly for that (eg https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi).

If you insist on manual design, download a sequence editor (eg Bioedit, https://bioedit.software.informer.com/7.2/), download sequences of your target from Genbank (https://www.ncbi.nlm.nih.gov/genbank/), align them, search for conserved regions with the desired melting temperature and distance from each other (depends on what type of PCR you plan to use). Once you are done, blast your sequences for specificity (https://www.ncbi.nlm.nih.gov/guide/howto/design-pcr-primers/) and test them on validated targets.

We use primer3 Web based software to design primers for PCR

https://primer3.ut.ee/

In order to satisfy some requirements, it is better to use online softwares dedicated to such task:

https://www.ncbi.nlm.nih.gov/tools/primer-blast/

https://www.genscript.com/tools/pcr-primers-designer

https://eurofinsgenomics.eu/en/ecom/tools/pcr-primer-design/

https://mfeprimer3.igenetech.com/spec

....

So the easiest way is to go through ncbi, which uses Primer3: https://www.ncbi.nlm.nih.gov/tools/primer-blast/

But if your doing it without software, just look for two 20 bp regions of ~50%GC separated by the desired size of your fragment, that have similar annealing temperatures of ~55C. Check out biohelper too:

https://www.yourbiohelper.com/

Dear Karyna Tarasova

You can find some guidelines at the following linmk on my blog, Proteocool

https://www.blogger.com/video.g?token=AD6v5dzxz85z69as_qBJCTiisMtVv4q-Q0ChLHJHqSwNRA_FWtuB9p36qi4nQxd4b3OB2cxClbV6BmNDgkIdwfDUZqcDdOrGBSsjcuGTQKHAIEJPIrzIbIxwHrRRfZQf_UG0xZwwe2I

i wish that it can be usefull to you.

good luck

Manuele

The easiest way to design the primers is to use online tool like. Primer3, eurofin genomics.

Just put your target sequence and mention the range of your target region you want to amplify.

However, if you want to design the primers manually you must keep these thing in mind.

1. Length of primer should be18 to 24 bp

2. GC content should be 40-60%

3. Melting temperature should be 50 - 60°C. 65 °C would be fine but exceeding 65 can cause secondary anealing.

4. Find the Tm of each primer either using this simple formula Tm = 2 X (A+T) + 4 X (G+C) - 2 or you can use online tool to calculate Tm.

5.Tm difference of both forward and reverse primer shloud not be more than 5°C.

6. Should be careful for GC Clamp. 3' end should have (2or 3)Gor C (with in last 5 bases) helpful in specific and strong binding. Avoide more than 3.

7.Make sure your primers are not complemantary to each other. Otherwise you will get primer dimers.

9. Make sure you make the complementary sequence of your reverse primer ie reverse complementary.

8. After designing your primer run an insilco PCR to check if your primers are working.(Online tool)

9. For safe side check the primers on oligo analyzer to check for hairpin loop or dimers.

Hope this helps

Good luck

I saw that some have already mentioned it here but that website provides the best and easiest way to resolve your issue. I highly recommend it. Give it a try if you have not yet.

https://www.ncbi.nlm.nih.gov/tools/primer-blast/

Manually, primers can be designed using simple MS Word or note pad and then validated on OligoCalc, where you can check the different criteria, like GC content, Tm, self complementarity etc.