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=== Removal of amplification adaptors === {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''2x rxn mix''' |- | ss amplicons||80.00||160.00 |- | USER||5.00||10.00 |- | Total||85.00||170.00 |} * Aliquot 85ul 2X, 37C for 2hr * Add 15.5ul of DpnII buffer and RE-DpnII oligos mix (mix 10.5ul of 10X DpnII buffer and 5ul of 100uM RE-DpnII oligo guide) toeach tube * 94C for 3min -> 37 C 3min * Add 5ul of DpnII, mix by pipetting 10 times on thermocycler * 37 C for 2 hr * Add 95 ul of USER enzyme mix (5ul USER, 10ul of 10X DpnII buffer, 80ul H2O * 37C for 2hr * Heat inactivate enzyme at 75C for 20min (optional) ''' PAGE verification before column purification '''<br> [[File:ZhangLab_2 2013-06-01 16hr 06min_Blueprint_PAGEverification.jpg| 200px]] Image descriptions: 1: ds amplicons 2: ss amplicons after lambda exonuclease digestion 3: USER digested ssDNA 4: USER -> DpNII digested ssDNA 5: USER -> DpnII -> USER digested ssDNA * Note: For lane 1-3, I took different volume of reaction to get the same amount of DNA in each lane to compare the efficiency of digestion by each enzyme * Purified with 4X QIAGEN column. * Eluted with 52ul EB buffer each (total volume = 208ul) * ssDNA Nanodrop = 31.7ng/ul, yield = 206ul * 31.7ng/ul = 6.53ug ''' PAGE size-selection '''<br> * Did PAGE size-selection in 3X 2D-well TBU gel, by loading ~2.2ug DNA/gel [[File:ZhangLab_2 2013-06-03 12hr 13min_PAGE-SS_Blueprint.jpg| 400px]] [[File:ZhangLab_2 2013-06-03 12hr 09min_PAGE-SS_Blueprint.jpg| 186px]] * Purified in 6X 1.5 mL lo-bind tube and resuspend each tube with 11ul H2O (total volume ~66ul) Image descriptions: ''' PAGE quantification of purified BSPP Blueprint '''<br> - I diluted 2ul of BSPP Bluprint probe with 38ul H2O then added 40ul of 2X tBU loading dye. - I then loaded different volume of dilute probe in 6% TBU gel (3, 4, 5, 6, 7, 8,& 10ul) - For LM ladder, I mixed 2ul of 1ug/ul ladder in total 20ul, and loaded 4, 6, & 8ul [[File:ZhangLab_2 2013-06-04 13hr 22min_BSPP_BP_PQ2.jpg| 350px]] Image descriptions: - The number is the loading volume of diluted probe (2ul in total 80ul). 3 = 0.075 ul 4 = 0.10 ul 5 = 0.125 ul 6 = 0.150 ul 7 = 0.175 ul 8 = 0.20 ul 10 = 0.25 ul - The concentration of the probe is very high, so the volume > 6ul (actual volume 0.15) gave a saturate signal * Total volume of probe 65ul, conc. by PAGE quantification: 48.2ng/ul, total amount = 65ul * 48.2ng/ul = 3.1ug (from total 48 PCR reaction) * For accuracy of probe concentration quantification, I currently use Qubit ssDNA assay. ''' 2013 - 06 - 03 ''' * Dinh received H1 gDNA passage #36 (conc. 149ng/ul, total amount ~8ug --> total volume ~ 52ul) from Belmonte's lab. * I re-quantify concentration by Nanodrop and Qubit dsDNA HS assay to confirm concentration and to compare the concentration between Nanodrop and Qubit. ** Nanodrop : '''100.36 ng/ul''', 260/280 = 2.07, 260/230 = 2.75 ** Qubit dsDNA HS assay: '''23 ng/ul''' (used 1ul of sample for quantification) * The concentration of H1 gDNA sample by Nanodrop and Qubit quantification is ~4X different between Nanodrop and Qubit. This might be the reason that I got very low yield after bisulfite conversion even I started with high amount of gDNA (1 - 1.2ug by Nanodrop before bisulfite conversion and measure bisulfite converted gDNA by Qubit ssDNA assay) . Note that in the past, I relied on the concentration by Nanodrop measurement since it's very convenient to measure many samples than Qubit. * I don't think it come from the calibration of machine since I used to measure standard DNA used for Qubit quantification (has very high purity) and Nanodrop, I got very close concentration by the two measurement. It should be the impurity in DNA that interfere 260nm absorbance. * In the future, I will use Qubit to measure gDNA concentration for BSPP experiment to reduce the inaccurate of DNA concentration due to the impurity of DNA samples. * It seemed like I will not have much gDNA to test for Blueprint project * Dr. Zhang suggested to not restrict to H1 DNA sample, but I can use any gDNA sample from HAPMAP plate since the 450K Illumina methylation array data are available and it should cover the assigned CpG sites. ---- Continued on: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-6-6]]
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