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'''Link to calendar:''' [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes]] == PCR amplification of circularized DNA captured with probes synthesized by LC Sciences (Library-free protocol) == * '''Aims:''' to be filled later: * Related lab note of BSPP captured with probes from LC Sciences: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-10-6 * I used the circularlized DNA captured by the probes synthesized by LC Sciences as the temples. ** PGP1-F (54C) ** PGP1-iPS (54C) {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''3.2x rxn mix''' |- | Circularized DNA template||2.00||0.00 |- | 10uM CA-2-FA.Indx.Sol||0.50||0.00 |- | 10uM CA-2-RA.Sol||0.50||1.60 |- | 2x KAPA SYBG MM||12.50||40.00 |- | H2O||9.50||30.40 |- | Total||25.00||72.00 |} * aliquot 22.5 ul, add 0.5ul CA-2-FA.Indx.Sol and 2ul of circularized DNA {| {{table}} | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Index''' |- | NTC||CA-2-FA.Ind7.Sol |- | PGP1-F 54C||CA-2-FA.Ind7.Sol |- | PGP1-iPS 54C||CA-2-FA.Ind45.Sol |} '''Program'''<br> 98°C 30sec -> (98°C 10sec -> 52°C 30sec -> 72°C 30sec) x8 cycles -> (98°C 10sec -> 72°C 30sec) x15 cycles -> 72°C 3 min -> 15°C hold<br><br> [[File:2012-04-22_Library-free-TestPCR_LC.png|600px]]<br> * 2 ul or 3ul of each sample were loaded in 6% TBE gel<br><br> [[File:ZhangLab_2 2012-04-22 19hr 07min_Library-free_LC_testPCR.png|300px]]<br> * From the picture, There were many bands appeared on the gel. However, I could still observe the band approximately 350 corresponding to the expected size of the product amplified by the long primers with barcode. For this experiment, I didn't expect to observe a single band of my product since in the previous experiment, I observed multiple bands when I did amplification after circularization with a short pair primer before barcoding and re-amplifying, on the gel as well. I should discuss with Dr. Zhang if we need to sequence these libraries to confirm our protocol, and if so is there any need to verify the libraries by colony PCR before sequencing or not. * Another thing I was thinking is that to increase the specificity, should I vary the PCR condition to get the optimum condition or not since the expected band seemed to be very faint? * 2012_04_23: Dr. Zhang suggested that it not necessary to optimize the PCR condition. I just do PAGE-size selection and pool the sequencing libraries. There is another sample from Baylor (RC-1) that was constructed the same way for SE sequencing and had a bad sequencing quality. He suspected that it might be due to the way we construct libraries for SE sequencing. Then he suggested to amplify for PE sequencing, so I need to order more barcoded primers. * '''Continued on:''' http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-4-23
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