Hi,
We are trying to do the emulsion PCR for screening of mutant alleles by following the procedure mentioned by Diehl et al (Nat Med. 2008 Sep; 14(9): 985–990) and Dressman et al ( PNAS July 22, 2003 vol. 100 no. 15 8817–8822). We are using Tegosoft DEC, ABIL WE 09 and mineral oil in the ratio of 73%, 7% and 20% respectively as mentioned in the protocol. 600 microL oil was first prepared by following the above ratio and it was layred over 150 microL aquous phase. A 5mm steel bead was added and the mixture was fractionated at 15Hz x 10s and 17Hz x 7s. The emulsion was divided into PCR tubes and PCR reaction was started. Up to this point it was OK but unfortunately the emulsion was breaking after 10-15 cycles. The oil and water phase is getting separated. We are getting cross reaction, may be due to this. We even tried different ratios of the above reagents as mentioned in the original patent. Nothing worked.
I have written to different persons regarding this but didn't get any reply.
If anybody can help in this matter it will be of great help.
Thanks.
Dear Ranjan,
If I underdstand correctly you want to do digital PCR.
Why not use the Biorad digital PCR system, or use other systems like Wafergen or the Quantstudio from Thermofisher. You could even do Next Generation sequencing on an Ion Torrent plarfrom.
Martin.
Actually this project is regarding emulsion PCR. I know about digital PCR.
Ranjan,
Maybe read this publucation first and find out if this is what you are looking for:
https://www.ncbi.nlm.nih.gov/pubmed/25744265
But this is in different language probably in French which I don't understand. Thanks. Can you help?
You are correct, did not realize this.
Try this link:
http://www.acgs.uk.com/media/1014434/10.40_-_02_-diagnostic_ctdna_analysis_of_egfr_in_non-small_cell_lung_cancer_angharad_williams__cardiff_acgs_2016.pdf
Thanks but this ppt mentioned about the pros and cons of ddPCR. Not the methodology. My problem is probably related with physical chemistry. I want to make a stable water in oil emulsion for PCR.
I have been through the same problem
I tried to make the emulsion using tween80/span80/triton X-100
Emulsion was made, but when PCR cycle is started the emulsions were broken btw the oil/water phases as you said
At that time, I concluded that the composition of the paper did not work or not proper for PCR
Recently, I saw the paper that uses the same emulsion composition
Article Simple paired heavy- and light-chain antibody repertoire seq...
Have you solved this problem?
I really wanna know the solution
Thanks
Thanks Kim for the update. We are still working on the project. But as I mentioned, the compositions mentions in the papers did not work for us. So we tried different ratios. Try using 1:2 and 1:3 combinations of water and oil.
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