Hello everyone
I am working on amplicons for species delimitation on corals.
My supervisor want me to make it through a 4 primers PCR on 4 loci (CR, ITS, ORF and ATPSbeta)
I have been trying for mounth to make this method works but it seems simply impossible
so basicly what i am doing is as follow
my MM is composed of
12.5 µl of green taq
1 µl of inner primer (F and R)
1 µl of barcodes (F and R)
8 µl of water
1.3 µl of DMSO
the cycle is as the screenshot i took (see pictures)
The PCR itself doesn't work, it oly work when i am diluting the barcodes. The more i dilute the more the PCR work (see image barcodes dillution). However if i dilute the barcodes, the PCR product isn't barcoded and it's impossible to retrieve any information from the sequencing.
I have been trying everything to make it work but i feel like there is no solution. Has anyone any advice to make this PCR work with barcoding ?.
I have also tried to separate the inner primer cycle (the 5 first cycle) from the barcodes cycle (the 30 last cycle), it makes the PCR work better but not that much.
i feel like the inner primer also amplificate the DNA in the last 30 cycle and that the barcodes are just amplificating the inner primer and doing dimer. I also tried to dilute the inner primer but then it doesn't work at all
Hi Olivier,
This is an interesting problem and fortunately, the one that I have been through. The PCR you are trying to do is also known as "Overlap-extension PCR"; a close-combat variant of "Nested PCR".
I would recommend to add the barcoding primers later in the cycle. Which means:
1. You increase the number of primary cycles (inner primer dependent) to 7-10, then introduce a hold at 25 (room temperature). Make sure your lid is still at 110 to avoid evaporation. However, be careful with the heated lid during the following steps.
2. Now at hold, you can open the lid and add the barcoding primers individually to your tubes. You can use a multichannel if you want. Since the volume would be lower, you can try diluting the primers to use a higher dispensing volume.
3. At this stage usually, I would also spike in 1ul of polymerase master mix; just to avoid resource exhaustion (Optional).
4. On the thermal cycler, you can skip to the next step to resume PCR.
I completely concur with you that this is a difficult PCR. Routinely done, but yet difficult. I am confident you will get good results with these simple changes. Let me know how it goes and feel free to DM me for more discussions.
hi Nikhil Hajirnis
thanks for your reply,
i don't think my PCR is an "OE PCR" as i am just trying to amplificate one amplicon and barcode it at the same time. So basically the inner primer possess an adapter on which the barcode can attach itself after the amplicon has been amplificated by the inner primer. However it seems that the barcode prioritize the inner primer over the amplicons and produce a large amount of dimer while not barcoding the amplicon.
i have already tried to separate the inner cycle and outer cycle but i will try to augment the number of inner cycle as you stated.
Is there any reason to have an hold step at 25°C ? i always put this step at 10°C and keep the well at low temperature using a cool rack.
Hi Olivier,
I agree that it's not exactly the OE PCR. I prefer 25 because it's room temperature. The reagents remain stable for that amount of it and since there's no additional denaturation condition like "initial denaturation" I prefer to keep it at that.
You might also try heating your primer mix to 80C and immediately put them in ice just before adding them to the reaction. This is because sometimes the primers might have higher probability of forming dimers. You will end up in separating them at higher temperatures and maintain them as such by immediately transferring them to ice. This can be done for long primers. But I am not sure how long your barcode primer is, so you can take a call accordingly.
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