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== Nicking endonuclease digestion == * Incubate 4ug of amplified amplicon with 10ul of Nt.AlwI (10U/ul) in total volume 200ul at 37C for 1hr (4x reactions) * Heat inactivate enzyme at 80C for 20 min * Add 10ul Nb.BsrDI (10U/ul) at 65C for 1 hr * 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 -->50.4ng/ul, yields = 15.1ug (why recovery rate so high > 85%?) * Before doing PAGE size selection to purify the 70nt band, I ran the gel to check if DNA nicking work well by loading the same amount of DNA before and after incubation with each enzyme at the same amount [[File:LC-probe-Nicking_2011_10_07.png| 280px]]<br> * A = amplified amplicon before nicking endonuclease digestion * B = After incubation with Nt.AlwI * C = After incubation with Nb. BsrDI * Note: The digestion was not very complete since I still see the partial digested band of ~ 90nt. I think that the condition using to nick amplified amplicon may not be very optimum since the amount of the input DNA is high and I try to minimize the volume of reaction. However in this experiment I have no time to optimize and I think that the expected band (~70nt or slightly lower) was a lot brighter than the incomplete digested band, so I continued to do PAGE size-selection to purify the 70nt band in 6% TBU gel. * PAGE quantification will be performed to check the efficiency of each enzyme by comparing the ratio of the intensity of each band. [[File:ZhangLab_2 2011-10-08 00hr 11min_PAGE-SS-1.jpg| 350px]][[File:ZhangLab_2 2011-10-08 00hr 15min_PAGE-SS-2.jpg| 380px]]<br> * I used the old TBU gel prepared 2 weeks and almost 1 month before, the resolution of the DNA was not very good. The gel prepare 2 week gave the a better resolution the 1 month prepared gel. Would recommend to use the fresh prepared gel or not > 2 weeks if there is no time restriction. * I incubated the gel eluted DNA overnight with EtOH, NaOAc, and Glycoblue at -80 --> will precipitate and quantify concentration on Saturday 08, 2011 * After EtOH precipatition and washing, resuspend the probe with 15ul each tube --> total volume 150ul * PAGE quantification in 6% TBU gel (mix 2ul of probe in total 80 u and load different volume to estimate DNA concemtration) [[File:LC-probe-PQ-1.png| 200px]]<br> * Final conc. = 4.10ng/ul --> total tields = 150ulx4.1ng/ul = 615ng (lower than expected)
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