Noi/NOTES/2012-5-16: Difference between revisions
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>Noi No edit summary |
>Noi No edit summary |
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#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'''<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)''' | ||
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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
- 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 #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