I agree with the above about accumulation of inhibitors. Sometimes this can be overcome by diluting the dna/cell suspension in your case ( less dna more amplification) and sometimes increasing the Mg concentration works if the inhibitors are removing Mg from the reaction mix

Multiplex PCR can be difficult by itself. Perhaps pooling different clones causes an accumulation of inhibiting factors. Does the PCR reaction work with only one colony? Maybe you can dilute the pool and adjust the number of cycles in your PCR accordingly. Changing your annealing temperature and extension time could also show to be beneficial. Hope it works, otherwise you probably need to do the extraction on your pools.

It looks like you have PCR inhibition.

1) is single PCR colony working?

2) Are you sure that by pooling colonies you are still in the conditions of single PCR colony in terms of mix concentration?

3) what exactly do you have? a smear? (could be an indication of too much DNA) or nothing at all?

I agree with the above about accumulation of inhibitors. Sometimes this can be overcome by diluting the dna/cell suspension in your case ( less dna more amplification) and sometimes increasing the Mg concentration works if the inhibitors are removing Mg from the reaction mix

Hello Farah.

It sounds that you need a complete lysis and denaturation procedure of the colonies first prior to completing washing away the lysed, denatured proteins and other cellular components.

Regards.

Dear Simon Strobbe,

Thanks A LOT for the reply

In our case of screening we need to do pooled multiplex PCR instead of one colony PCR due to the high number of clones that we have and a good number of genes looking behind. Still I can not try single colony PCR because I do not know the exact clone which has my desired gene (It is the 3D pooled PCR screening method).

May be the idea of diluting the Template and changing annealing temperature is a good one, I have to try.

But why do you think that the extension time needs to be changed?

Thanks again

Dear Marie Wattenhofer-Donzé

Thank you very much for your reply

1) I am not able to do single colony PCR because the aim is to try a lot of clones in same time to know where is my gene within.

2) I think this method has been used before but I do not have information about how many of the bacteria I have to use so I just pool few micro litters from each and pool into one tube to take 2ul for PCR.

3) I had very pail band of less than 100bp, my genes size is at least 500bp

Dear Paul Rutland

Thank you so much for the answer

In my PCR reaction 1.5mM of MgCl2 is there, I I want to try to add I think I can try up to 5mM (Please correct if I am wrong)

But concerning the template of colony, I am not sure about dilutions that I need to try ( any suggestions )

Very cordially!

Dear Sang Ho Lee

Thank you Prof for the reply,

I think your suggestion is my plan B, because I have a big library of clones , now I am trying to optimize my colony PCR if no results I may have to do as you recommended.

Thanks again

Dear Farah,

I think that you have to titrate a representative sample because the Mg and inhibitor concentrations are linked. I would use 3mM mg and dilutions 1:1, 1:2, 1:4 and 1:8 and if one dilution works then use that dilution and Mg from 1.5 to 5mM in increments of 0.5. If you have primer dimer then decrease the amount of primer and try increasing the annealing temperature. Check that the negative control is really negative as pcr contamination of primer dimer can stop a reaction from workinh by removing primer rapidly from the reaction mix

Dear Farah Fadwa Ben belgacem,

Glad to hear you consider my suggestions useful.

I was just thinking that the inhibiting factors might hinder your extension. Therefore i considered a prolonged extension might minimize this effect.

you refer above to a pale band of less than 100bp. This could be primer dimer.is it in your negative control.If it is PD then you can try using less primer or a hot start enzyme.It may look weak but it is short so does not intercalate much ETBR and there could really be a lot of it and it could be removing primer from your reaction mix

@ Paul Rutland Good Question

Regards

Thanks again for all of you.

Dear Paul Rutland,

Actually I did not include negative control in my PCR trial. If I am not mistaken, you do mean one PCR reaction without template, isn't?

Hi Farah

I agree with Paul. Your 100 pb looks like dimer primers. and yes the good control is your PCR but without template.

another question for you. How do you dilute your colonies? LB is indeed a strong PCR inhibitor. did you try just for test to do the same on one colony only ? (just to validate your protocol)

In principle, the greater number of colonies in multiplex PCR, the more difficult to optimize reaction conditions. You can adopt the conditions of the successful reaction and use it with the same proportions for other colonies. Then you may fine-tune annealing temperature. One more thing, low molecular size often refers to dimer.

Dear @Marie Wattenhofer-Donzé

Thanks for the reply,

yes I usually use same bacterial media to dilute in my case LB, so what to use instead?

As I do not know which clone within a big library has my target gene, I can not optimize my PCR using one clone I have to use pooled clones.

Dear @Ayoob Obaid Alfalahi

Thank you so much,

Actually I am trying to use the option of gradient PCR in the thermocycler PCR, using interval of 5 degrees above and 5 below annealing temperature, hopefully it will work.

All the best!