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

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Line 12: Line 12:
  #3-V6         132.20         15.86                 15.86
  #3-V6         132.20         15.86                 15.86
  #4-V6         101.70         12.20                 12.20
  #4-V6         101.70         12.20                 12.20
'''DpnII digestion reaction set up'''
 
{| {{table}} border = 1
'''DpnII digestion reaction set up'''<br>
  {| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Probe IDs'''
| align="center" style="background:#f0f0f0;"|'''Probe IDs'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)'''
Line 41: Line 42:
  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'''
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 -> Qiaquick column purification

Revision as of 23:43, 16 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
#2-V4	        125.10	         15.01	                12.00
#3-V4	        110.10	         13.21	                13.20
#4-V4	        133.80	         16.06	                 8.00
#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 -> Qiaquick column purification