I explain, i'm doing PCR on Chlamydia trachomatis. The PCR on strains works fine, i got large bands on gel, and when i'm sequencing it, the result is perfect.
Then i had to do the PCR on patients samples, it's where my problem began, for all my patients I got bad or none amplification (which is strange because they all came up positive with another method automated Taqman realtime PCR that we use in routine diagnosis), except for my positive control, which is a positive patient 16 ct that i diluted in H20 + genomic human DNA.
I'm feeling like maybe the human DNA in the water is somehow enhancing my PCR and make it work perfectly for patients, for strains there is no need of this since there's so much pure material that it probably works on his own.
What do you think ? I will try to dilute my patients with the same h20 DNA i used for my control positive, maybe the genomic DNA works as a carrier or something like this. I also will try to up my hybridation time and my elongation time a bit, to see if there's any improvement.
If you have any more ideas, i will be pleased to read them.
Cheers
Hi Marc-Antoine,
are you using the same primers and enzyme in the PCR as you use in the RT pcr. Some enzymes are more fussy than others about contaminants. Also taqman is very sensitive...it may be that you just need more cycles of pCR. In all PCR related questions it is best to let people know the enzyme and cycling conditions and ,ideally, primer sequences to get the best advice otherwise everyone is guessing a bit.
good luck
paul
I am not aware of this but if you could culture your bacteria and then go for your diagnostics ... does it will work?
@Paul Rutland : It's a premix from ABI you can use it for RT and Classic PCR (AmpliTaq Gold Fast Master MIX) I'm doing 40 cycles in classic PCR which is already more cycles than in Taqman. I Also tried with an old mix and it get some bands even with patients, so there's a problem with my ABI mix, but it is so much faster and sensitive than the old mix i need to go with it.
@Hossein Jamali : It's what i will test today, but if it works, i really don't know why the fact of making dilutions with Dna water could change anything? Maybe Dna is acting like a carrier or supporting the reaction?
@Prakash Chandra : We are doing both in our laboratory and for large spectre detection in emergency, sometimes you dont have time to grow cultures from patients, or sometimes the cultures just don't grow (intracellular germs, complicated germs). So I have to make this PCR works both on cultures strains and also directly from patients samples.
Thanks for you answers, i really appreciate your help.
I added Dna + H20 when i did my dilution and it worked like i thought.
Then I tried to increase the time of hybridation from 3s to 5s and elongation from 12s to 15s
Works perfectly, got large bands, my problem is solved.
well done Marc-antoine its always good to hear things are working and knowing the reason why
best wishes
paul
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