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== Nicking endonuclease digestion == * Incubate ~3.2ug of amplified amplicon with 10ul of Nt.AlwI (10U/ul) in total volume 200ul (in NEBuffer2) at 37C for 1hr (4 x reactions) ** Each tube, 36ul 2nd round amplicons (89.6ng/ul), 20ul 10X NEBuffer2, 10ul Nt.AlwI, 134ul H2O * Heat inactivate enzyme at 80C for 20 min * Add 10ul Nb.BsrDI (10U/ul) at 65C for 1 hr * Before doing column purification, I ran the gel to check if DNA nicking work well by loading DNA before and after incubation with each nicking enzymes in TBU gel [[File:ZhangLab_2 2013-03-09 13hr 39min_Nicking.jpg| 150px]] I loaded 1ul of nicking enzyme reaction mix '''U'''= undigested '''Nt''' = Nt.AlwI '''Nt/Nb''' = Nt.AlwI & Nb.BsrDI * Note: >90% of the oligos were completely nicked by the two nicking enzymes. One thing I am concerned is the size of probes is quite long, so the 210, 190 and 170nt stay very close in 6% TBU gel. I may need to run a little longer for 5 or 10 minute to get a better resolution. * Purify with 6 Qiaquick colums, elute with 50ul EB buffer each --> total volume 300ul (Note: use more Qiaquick columns since the volume of of reaction was large, total 800ul and doing multiple pass of DNA through the column didn't help to recover DNA very well) * Measure the conc. of nicked DNA with Nanodrop --> 37.8 ng/ul total volume 6*50 = 300ul, yields = 11.34 ug (recovery rate ~ 84.4%) * I did PAGE size-selection by loading ~1.8ug (to purify in 6x 6% TBU gels and prevent overloaded because the bands are very close). Since the 10bp ladder resolution around 150-higher is not very good, I loaded nicked DNA as marker in small well since I did PAGE verification before as shown in the image above. [[File:ZhangLab_2 2013-03-09 19hr 22min_PAGE_SS-1e.png| 400px]] [[File:ZhangLab_2 2013-03-09 21hr 01min_PAGE_SS-2.jpg| 410px]] [[File:ZhangLab_2 2013-03-09 21hr 01min_PAGE_SS-3.jpg| 400px]] * EtOH precipitate and resuspend functional probes in total volume 260ul. * Perform PAGE quantification in 6% TBU gel (mix 2ul of probes in total volume 100ul and load different volume -> 2, 4, 6, 8, and 10ul) * Actual loading volume is 0.04, 0.08, 0.12, 0.16 and 0.2 ul [[File:2013_03_10_PQ_12K_InsituProbes.png| 300px]] * From the gel image, it seemed like I loaded too small volume and the intensities of ladder were not very reliable. The reliable volume is ~ 0.16 and 0.20 ul of actual volume of the probes ~3.7ng/ul. So the total yields ~ 3.7ng/ul * 260ul ~ 962ng. * However, I might re-quantify or do Qubit ssDNA assay to confirm concentration before start capturing * Note: Since I resuspend the probes in too large volume, I concentrate using Qiaquick column, elute in total volume ~62ul, re-quantify conc. ~22ng/ul (by column purification I lose ~10-15% probes) * Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-12
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