Noi/NOTES/2012-5-16: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Noi
>Noi
No edit summary
Line 40: Line 40:
| #4-V6||101.70||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.
  - 37C 30min -> 65C 20min -> Qiaquick column purification (also combine the samples used for DpnII digestion trial.
  Measure concentration by Nanodrop and calculate yield
  - Measure concentration by Nanodrop and calculate yield
   
   
  '''DpnII digested DNA conc. and yields'''
  '''DpnII digested DNA conc. and yields'''
Line 80: Line 80:
|}   
|}   
      
      
  37C 1h -> 75C 10min -> EtOH precipitate
  - 37C 1h -> 75C 10min -> EtOH precipitate
  I took 0.5ul of digested DNA before precipitation and run check in 6% TBE-urea gel   
  - 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]]     
  [[File:ZhangLab_2 2012-05-16 19hr 56min_DpnII-Exo.jpg| 400px]]     
Line 88: Line 88:
  1,2,3, and 4 = oligo set #
  1,2,3, and 4 = oligo set #
  Anyway, PAGE doesn't tell if exonuclease completely cleave or not.
  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

Revision as of 17:10, 17 May 2012

Probe production for the HiResChrPaint project

 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