Dear Haider,

It is not easy to establish a new multiplex PCR (extra long DNA amplification with 4500 bp amplifcates)!

Did you check all possible primer combinations in separated single PCR's?

Did these single PCR's all work very well?

Use all single PCR's the same forward or the same reverse primer?

What are the concentrations of the primers?

What is your PCR protocol?

Best regards,

Thomas

I would like to see agel with just non amplified dna in the well in case the large dna is actually protein. Also dna can remove MG from solution so it is possible that 19primer sets remove too much mg so the enzyme cannot work, You could try a MG gradient as well as less dna. If this were my problem I would test all primer sets in one reaction each and only then try 4 primer sets at a time and if this works then add more primer sets for as long as the pcr keeps working

Dear Thomas M.C. Binder

Thank you for your comments

I did PCR reaction with single primers and their resolution in gel is well.

In pool of primers there are two group of primers that are working with 2 and 3 reverse primers and 1 forward, I am working with polymorphic targets therefore, I include in these reverse primers to cover the most number possible targets of population studied, this multiplex primer was designed with less number of primers however now I am trying to cover more sites so I decide to increase the primer numbers.

About the concentration of primers I have attached a concentration table of the primers that I use.

the PCR protocol is 95°C 10 min, (95°C 11 sec- 63°C 1 min-70°C 5 min)*30 cycles, 4°C hold, following the company specifications for this polymerase

Best regards,

Haider

Paul Rutland thank you so much for your suggestions, I really appreciate it, and I will apply it, regarding the agarosa gel with just the sample, I have attached the respective gel.

Dear Haider,

This seems do be a little bit difficult.

Could you give me graphical scheme were your primers bind to the target gene? (You must not give me the name of the target gene.)

Do all forward and all reverse primer bind to the same positions of the gene?

How many amplfication products you can get with one sample (one amplicon, two or more)?

Did all amplicons have the same size, of about 4500 bp?

What are the differences between all forward and all reverse primers?

or: How similar are all forward and all reverse primers?

Only one base or more bases on primer 3'-end?

To give an example:

Could primer 1F with primer 2R, 3R, 4R, 5R, 6R, 7R or 8R give amplicons?

and so on ...

Best regards,

Thomas

Hi Haider,

If that gel has the amount of dna that goes into each pcr then I have 2 worries. 1 there is too much dna....25ng of good dna should be enough but you seem to be using much more and this can inhibit a pcr by removing Mg ions. or 2 the dna is contaminated with ptotein giving a large size band and the protein may be inhibiting the pcr. An OD 260/280 should be about 1.8 and I think that as well as running a Mg gradient you should titrate the dna so set up pcrs with 1/2, 1/4, 1/8 and 1/16 th the amount of dna that you are using and run 35 cycles of the pcr. Sometimes less dna means less pcr inhibitor and the pcr runs better with less dna but no inhibition

Dear Thomas M.C. Binder ,

Thank you very much for your advice, I really appreciate your help.

the primers bind the same position of the genome, however, these amplicons are not always same, therefore, the product length sometimes is different, but the variation is not significant.

just one pair of primers can show two bands, but it is very near of the expected band, respect of amplicons size, they are different, however, 3 have almost the same length (around 3kb).

only in 2 targets I am using the system of 1 F and 2,3 R, and those F not share similarities, I have include the scheme of primers, today I mix the primers using the suggestion of Paul Rutland in two different mixes, since I know the problematic primers, I know that the primers 2F and the first 2R is binding well, also the pair 3F and the first 3R, also I added the respective picture and primers resolution in a gel of 0.5%.

the second 2R primer, second 3R and third 3R are in not conservated, in my experiment those regions are important for samples that share these sequences, furthermore, the first 2R and the first 3R is in conservated sequences.

Thank you so much again for your cooperation. I really appreciate your valuable advice.

Best regards,

Haider

Hi Paul Rutland ,

thank you very much for your suggestions, I am currently trying to add a primer else to amplify the target region of the second and third Reverse of group 2 and 3 (primers that are not conservated in the population studied), in order to make its amplification easier, I am also going to try to use 25 ng of DNA, I usually use 50 ng, the previous gel had loaded 100 ng, thank you very much for your advice, I really appreciate it.

best regards,

Haider