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Old 02-16-2017, 01:00 PM   #1
meribur
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Default Library PCR cycle optimisation

Hi there,

This is probably a very naive question, but how can I test the number of cycles required to do library enrichment, if I have a limited amount of material- enough for just one shot.
Normally, I would do several PCRs, with range of cycle from 8-15, but here I only have enough for one tube/one PCR

Thanks for your help
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Old 02-16-2017, 06:13 PM   #2
nucacidhunter
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As a background, if by optimisation you mean yield then you should consider quantity of library required for downstream steps including QC, quantification and sequencing. Generally, 30 ul of 5nM library is sufficient for all those steps and repeat runs on Illumina systems. You can calculate molar concentration back to required ng of library and the yield you would require from PCR after clean up allowing 30% loss during clean up.

Options for this case:
1- using another similar DNA source (quantity, extraction method, ) as proxy for optimisation
2- taking 1ul from PCR after 8 cycles and running on ScreenTape or a similar method to look for yield and profile. You should be able to calculate additional required cycles by approximately doubling yield with each cycle and rechecking again before clean up. This is the easiest way if you have access to a TapeStation or a Bioanalyser. If input DNA quantity is high it should be taken into account when calculating additional cycles.
3- Using a real time PCR machine by adapting protocol from the following link (page 16) or similar commercial kits:

http://rubicongenomics.com/wp-conten...ed-9_16_16.pdf
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Old 02-17-2017, 12:10 PM   #3
meribur
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Thank you for your reply, I have one more trivial question

I think that for me the most easy thing to do would be to, as you suggested, try 8 cycles and take 1ul out to check yield. If I do so, and for example decide I need another two cycles, would I then re-PCR this adding more of master mix? In normal PCR, when I need to amplify more of my product, I use the whole PCR and add more of polymerase, buffer, primers, but for this here I am not quite sure..

thank you and sorry for being ignorant..
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Old 02-17-2017, 01:57 PM   #4
nucacidhunter
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PCR need to be run as 8 full cycle s(including final extension) then kept cool while the analysis is being done. Additional cycles can be done on the same reaction without adding any reagent and skipping initial heating if polymerase was a hot start type.
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