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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] == Probe production for the HiResChrPaint project == * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-14 * Since Dr. Zhang suggested that about 20% of the oligos having AP2 part should be fine. I will continue to digest the rest of amplified amplicons with DpnII and column purify Probe IDs Conc. (ng/ul) Yields in 120ul (ug) Left over after trial (ug) #1-V4 83.70 10.04 5.00 after combining with the tube digested a day before total amount should be 10ug #2-V4 125.10 15.01 12.00 after combining with the tube digested a day before total amount should be 15ug #3-V4 110.10 13.21 13.20 #4-V4 133.80 16.06 8.00 after combining with the tube digested a day before total amount should be 16ug #1-V6 122.50 14.70 14.70 #2-V6 106.20 12.74 12.74 #3-V6 132.20 15.86 15.86 #4-V6 101.70 12.20 12.20 '''DpnII digestion reaction set up'''<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Probe IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="background:#f0f0f0;"|'''10X DpnII Buffer''' | align="center" style="background:#f0f0f0;"|'''DpnII''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' | align="center" style="background:#f0f0f0;"|'''# of tube''' |- | #1-V4||83.70||59.74||22.26||10||8||100.00||1 |- | #2-V4||125.10||47.96||34.04||10||8||100.00||2 |- | #3-V4||110.10||59.95||22.05||10||8||100.00||2 |- | #4-V4||133.80||59.79||22.21||10||8||100.00||1 |- | #1-V6||122.50||60.00||22.00||10||8||100.00||2 |- | #2-V6||106.20||59.98||22.02||10||8||100.00||2 |- | #3-V6||132.20||59.98||22.02||10||8||100.00||2 |- | #4-V6||101.70||59.98||22.02||10||8||100.00||2 |} - 37C 30min -> 65C 20min -> Qiaquick column purification (also combine the samples used for DpnII digestion trial. - Measure concentration by Nanodrop and calculate yield '''DpnII digested DNA conc. and yields''' Probe IDs Conc. (ng/ul) Yields in 80ul (ug) #1-V4 86.10 6.89 #2-V4 137.40 10.99 #3-V4 110.90 8.87 #4-V4 134.00 10.72 #1-V6 137.00 10.96 #2-V6 124.40 9.95 #3-V6 155.70 12.46 #4-V6 113.60 9.09 Total yields of DpnII digested DNA are between ~7ug-12ug. I will set up exonuclease digestion in volume 100ul with 100U of lambda exonuclease '''Lambda exonuclease digestion reaction set up'''<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Probe IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''DpnII digested DNA (ul)''' | align="center" style="background:#f0f0f0;"|'''10X Lambda exo buffer''' | align="center" style="background:#f0f0f0;"|'''Lambda exo (10U/ul)''' |- | #1-V4||86.10||80.00||10.00||10.00 |- | #2-V4||137.40||80.00||10.00||10.00 |- | #3-V4||110.90||80.00||10.00||10.00 |- | #4-V4||134.00||80.00||10.00||10.00 |- | #1-V6||137.00||80.00||10.00||10.00 |- | #2-V6||124.40||80.00||10.00||10.00 |- | #3-V6||155.70||80.00||10.00||10.00 |- | #4-V6||113.60||80.00||10.00||10.00 |} - 37C 1h -> 75C 10min -> EtOH precipitate - I took 0.5ul of digested DNA before precipitation and run check in 6% TBE-urea gel [[File:ZhangLab_2 2012-05-16 19hr 56min_DpnII-Exo.jpg| 400px]] U=undigested DNA 1,2,3, and 4 = oligo set # Anyway, PAGE doesn't tell if exonuclease completely cleave or not. - I precipitated DpnII/Exo oligos because in the following step of probe labeling with ARESβ’ DNA Labeling Kits (Invitrogen) it requires approx.1-5ug of DNA in limited volume 5ul. - From total 100ul Exo digestion reaction I added more H2O 200 ul (total volume 300ul), 1ul Glycoblue, 30ul 3M NaoAc pH5.5 and 900ul 100% EtOH. -80C O/N - Resuspend with 7ul H2O '''PAGE quantification in 6% TBU gel''' Dilute 100x (1ul in total 100ul) of purified ssDNA --> load 3ul --> Actual volume in each well = 0.03ul [[File:ZhangLab_2 2012-07-11 17hr 58min_PQ_ChrPaint_1.jpg| 400px]] {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Total amount in 6ul (ug)''' |- | #1-V4||278.49||1.67 |- | #2-V4||432.80||2.60 |- | #3-V4||359.76||2.16 |- | #4-V4||438.99||2.63 |- | #1-V6||425.30||2.55 |- | #2-V6||427.85||2.57 |- | #3-V6||426.04||2.56 |- | #4-V6||306.88||1.84 |} * Note: I quantified only the completely DpnII/Exo digested ssDNA * The ssDNAs were handed to Dr. Zhang for dye coupling on 2012_07_12: http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/HiResChrPaint/2012-7-12
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