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

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* Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-14
* 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
* 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 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| 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

Latest revision as of 16:25, 14 July 2012

Probe production for the HiResChrPaint project[edit]

 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