Golla Ramanjaneyulu You can get help in designing primers from NCBI using primer-BLAST. Various tools and softwares are also available for designing primers.

You have to keep following things in mind (you can modify according to your criteria):

1. The primer sequence is annealing to unique sequences that flank the specific target and not to other non-target regions in the sample.

2. Having 18 to 24 nucleotides.

3. Primers having 40-60% GC content of the template.

4. Avoid complementary sequences at the 3′ end of the primer pair. Avoid GC-rich 3′ end. Also avoid mismatches with the target at 3′ end.

5. Design primer having G or C residues in central regions and 5′ end.

6. Avoid sequences which might have the potential in forming internal secondary structures.

Hope it helps.

A lot depends on the structure of the gene and why you want to amplify it. If it is a single exon gene then you have the choice of amplifying with overlapping amplimers or amplifying the whole exon and sequencing with overlapping sequencing primers within the amplimer. What is the problem with using primer3 or similar software....is the gene very low GC or high GC and what do you want to do after amplifying the gene?

In simple terms, any pair of complementary sequences will work as a primer. There is a number of rules for effective primer design such as G/C content, melting temperature, lack of complementary fragments within the primers. You can read about it here https://www.addgene.org/protocols/primer-design/ in more detail. There is a number of software tool that can generate the primer pair from any given sequence including Snapgene, already mentioned primer3 and number of web-based tools that can be found by google search "primer design". You need to provide more information about what do you mean by the "gene does not follow any criteria in designing primers".

It definitely needs to be a 2 step or 3 step amplification starting from a larger fragment to amplify followed by inner primers of desired sequence/ size. This eliminates the possibility of off-target binding. To eliminate poor binding of primers (low GC), since you have to keep the annealing temp in a certain range, just try/ test few different ones to get the best amplification.

Some amplicons have more problems than the others but in my view you may use a couple of primer pairs and try using a couple of different enzymes.

Such as use a 20 -30 bse long primer pairs. You may choose two primers of any length between these. BUt do make sure two things.

1. That the Tm of both primes in a pair do not differ more than 3 degrees.

2. definitely try to end your primers at have G or C at the 3 prime end- and you may vary the primer length to achieve this such as if you find AAAG at positions 21-24 then make your primer 24 bases long which includes AAAG but if you find the sequence to be AGAG at positions 21-24, then you may end the primer at 22 bases since you already got a G at the end.

Then choose your enzymes.

My favorites are the Phusion and One taq from NEB.

I have always found that if I have difficulty with one enzyme then the other works and vice versa.

Note: The Tm of the primer depends on the buffer conditions which are unique for different enzymes- thus use the appropriate Tm calculator for calculating the Tm.

https://tmcalculator.neb.com/#!/main

follow

• Length of 18-24 bases
• 40-60% G/C content
• Start and end with 1-2 G/C pairs
• Melting temperature (Tm) of 50-60°C
• Primer pairs should have a Tm within 5°C of each other
• Primer pairs should not have complementary regions Note: If you will be including a restriction site at the 5’ end of your primer, note that a 3-6 base pair "clamp" should be added upstream in order for the enzyme to cleave efficiently (e.g. GCGGCG-restriction site-your sequence)

Primers are always specified 5' to 3', left to right. ...

Primers for PCR and sequencing should be between 18 to 30 nucleotides in length.

Primers for PCR and sequencing should have a GC content between 40 and 60%, with the 3′ of a primer ending in C or G to promote binding

Try using Primer 3. It’s online and free.

You just need to input the sequence of the gene of interest. After running, it will give a lot of possible primer. Just use the common criteria in choosing a good primer.

1. Temp is around 50-60C

2. High GC content

3. Temp of forward and reverse primer is near to each other.

If you are not an expert in molecular biology, somebody expert may support you for this case. Nevertheless, if you want to do it selfishly, I highly recommend you to use PerlPrimer, an excellent user-friendly and versatile PCR primer design software. You can get it through the following link:

http://perlprimer.sourceforge.net/

Golla Ramanjaneyulu You can get help in designing primers from NCBI using primer-BLAST. Various tools and softwares are also available for designing primers.

You have to keep following things in mind (you can modify according to your criteria):

1. The primer sequence is annealing to unique sequences that flank the specific target and not to other non-target regions in the sample.

2. Having 18 to 24 nucleotides.

3. Primers having 40-60% GC content of the template.

4. Avoid complementary sequences at the 3′ end of the primer pair. Avoid GC-rich 3′ end. Also avoid mismatches with the target at 3′ end.

5. Design primer having G or C residues in central regions and 5′ end.

6. Avoid sequences which might have the potential in forming internal secondary structures.

Hope it helps.

First, you have to retrieve the full gene sequence, then,use the primer3 or NCBI BALST to get many proposed primers,

- Main criteria you have to follow, choose the primer pairs that flank your target fragment, not another nono specific one

- GC% should be 50-60%,

- avoid primers with the probability to make dimers or hair pins.

- then make insilico PCR to make sure the working of the obtained primer pairs before primer ordering.

Dear Golla

i do not undestand what do you mean with: " does not follow any criteria in designing primers how to proceed with that sequence"

however in case it can help you i would like share with you my blog

ProteoCool https://proteocool.blogspot.com/p/cloning.html

where are PAGE1 you can find 2 video regarding Primer desing for gene cloning.

good luck

MAnuele

From my experience, I would propse "Primer Design | Custom DNA Oligos‎" tools from Thermo Fisher (see https://www.thermofisher.com/de/de/home/life-science/oligonucleotides-primers-probes-genes.html?gclid=EAIaIQobChMIi9e6-6yN6gIVx7YYCh0fJA-eEAAYAiAAEgJ4qPD_BwE&s_kwcid=AL!3652!3!264260528307!e!!g!!primer%20design&ef_id=EAIaIQobChMIi9e6-6yN6gIVx7YYCh0fJA-eEAAYAiAAEgJ4qPD_BwE:G:s&s_kwcid=AL!3652!3!264260528307!e!!g!!primer%20design&cid=bid_mol_pch_r01_co_cp1358_pjt0000_bid00000_0se_gaw_ta_pur_con).

this website can help

http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi