Noi/NOTES/2012-5-16
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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
Probe IDs | Conc. (ng/ul) | Volume (ul) | H2O (ul) | 10X DpnII Buffer | DpnII | Total volume (ul) | # 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
Probe IDs | Conc. (ng/ul) | DpnII digested DNA (ul) | 10X Lambda exo buffer | 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 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 ARES™ 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
Probe IDs | Conc. (ng/ul) | 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 hand to Dr. Zhang for dye coupling on 2012_07_12: http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/HiResChrPaint/2012-7-12