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=== E. ChIP-Seq Library Amplification by PCR === * I would initially do a quick qPCR to test the PCR cycle number {| {{table}} border = 1 | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total 100ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total 15ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''8.5 rxn''' |- | SeqAmp PCR Buffer (2X)||50.00||7.50||63.75 |- | Forward PCR Primer (12.5uM)||2.00||0.30||2.55 |- | Reverse PCR Primer (12.5uM, R3)||2.00||0.30||2.55 |- | 50X SYBR||0.40||0.06||0.51 |- | SeqAmp DNA Polymerase||2.00||0.30||2.55 |- | Total||56.40||8.46|| |} :- Aliquot 8.46ul + 6.6ul of DNA template :- Split 4.5ul to 3X 8-tube strip :- '''ChIP-PCR:''' 94C, 1min -->[98C, 15s -> 55C, 15s, 68C, 30s] 11X only for Epi hg19 UMR, 15X, 16X, 18X --> 4C hold '''Note:''' I used REalplex (Eppendorf) for amplification<br> :- Load all 4.5ul PCR product in 6% TBE gel. * Note: To save the TBE gel, I did not load all PCR cycles. Since 10ng of Hg19 LMS v1 shoot up very quick at 11 cycle as recommended by the kit, I stopped it earlier. [[File:ZhangLab_2 2014-10-29 17hr 52min_DNASMRT-11XEpi-16x.jpg| 450px]] * : over-amplified ** I made a wrong labeling. It should be UMR instead of LMS [[File:ZhangLab_2 2014-10-29 17hr 52min_DNASMRT-18x.jpg|400px]] * From PAGE verification, I saw the PCR product size of Hg19 LMS v1 between ~180-almost 300bp. I really had no idea what is the actual size of sequencing library should be as I did not know the actual length of the probe. I believed that the experiment worked since I did not see this signal in NTC reaction. I continued to amplify the left over template (37.4ul) for 12 cycles. {| {{table}} border = 1 | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total 100ul''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total 85ul''' |- | SeqAmp PCR Buffer (2X)||50.00||42.50 |- | Forward PCR Primer (12.5uM)||2.00||1.70 |- | Reverse PCR Primer (12.5uM, R10)||2.00||1.70 |- | 50X SYBR||0.40||0.34 |- | SeqAmp DNA Polymerase||2.00||1.70 |- | Total||56.40||47.94 |} :- '''ChIP-PCR:''' 94C, 1min -->[98C, 15s -> 55C, 15s, 68C, 30s] X12 --> 4C hold :- To save all product above 100bp, I use a little high ratio of AMPure bead (1.8X) to purify the PCR product and eluted with 30ul H2O :- I took 4ul for PAGE verification. I also included 2ul of diluted original probes from Nimblegen (total amount ~11.7ng) to verify the size in 6% TBU gel [[File:ZhangLab_2 2014-10-29 20hr 59min_TBU-verify_Epi-LMS-v1-ori-seqlib.jpg|180px]] * I made a wrong labeling. It should be UMR instead of LMS * It look like I still have some background of the band around 150bp and primer dimers. To may need to do one more round of bead purification to exclude these background.
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