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Old 01-11-2018, 01:20 AM   #1
mastermix
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Default 4C Question

I did my first 4C libraries and it seem to work fine. The protocol is long but easy. The principle is quite clear to me but I have a question to the final step. I start with 10 million cells and potentially expect 20 Million interaction between my viewpoint and other DNA sequences. Iím very aware that with the two digest and 2 ligation I will end up with a super pure efficiency and just a view circular DNAs containing my viewpoint. In addition they have to be small enough to be amplified by PCR.
My Idea for the final library amplification including Barcodes and Linker for the Illumina platform would be to take a lot of DNA after the second ligation (to ensure to get the most complexity of different interaction) and just do the minimum of cycles to ensure that shorter sequences will not overwhelm longer during the PCR. But the protocol sais I should do a 16 PCR with 30Cycles which reaches the plato of the reaction and pool them afterwords. Since just some pg of this DNA is used to load on the Illumina Flow-cell. Iím a bit pusselnd why 4C follows this stategie. I would be happy if someone knows it or has a useful link to the answer.
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Old 01-11-2018, 05:08 AM   #2
Asashoryu
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Indeed there are 20 million possible interactions, but in reality the number is much smaller. The reason for large volumes and large number of cycles is therefore to have more specific PCR product (which starts from very few molecules) compared to input DNA. Otherwise you would be mostly quantifying your non-amplified input. The reaction itself is sometimes still not even saturated at 30 cycles.
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Old 01-11-2018, 05:40 AM   #3
mastermix
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Thanks so much for your answer! If the only problem is the library quantification, it would be better to measure PCRed DNA fragments having the Illumina adapters by KAPA qPCR after some few PCR cycles. The non amplified DNA input should not interfere with the sequencing since it contains no adapter sequence needed to bind to the flow-cell or?
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