Dinh:Protocols/Probes Prep Nov17
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November 17, 2009[edit]
Step 1: Real Time PCR Probe Amplification[edit]
1. Prepare 96 Reactions (12x or 1.2ml per tube):
Reagent | Final Conc. | Vol (1x) | Vol (100x) |
---|---|---|---|
ddH20 | 49ul | 4.9ml | |
NEB Tag 2x Master Mix | 1x | 50ul | 5.0ml |
AP1V4IU (100uM) | 0.3uM | 0.3ul | 30ul |
AP2V4 (100uM) | 0.3uM | 0.3ul | 30ul |
SYBR Green (50x) | 0.2x | 0.4ul | 40ul |
Template CpG30k (20nM) | 33pg | 0.05ul | 5.0ul |
Total | 100.05ul | 10.005ml |
2. Program
94C - 2 min 94C - 30 s 60C - 1 min 72C - 45 s Plate Read Cycle to 2 for 19 more times 72C - 5 min 15C - forever end
3. Stopped at 20 cycles.
November 18, 2009[edit]
Step 2: Ethanol Precipitation[edit]
1. In Solution Basin (55ml) Add:
NaOAc (3M), 960 ul GlycoBlue, 32ul Ethanol 100%, 24ml
2. Pool all reactions from 1 plate into Solution Basin (55ml) 3. For each plate, transfer equally to 8x15 ml tube. Store at -70C for more than 20 min 4. Turn on centrifuge (might need to switch the "swirlers") 5. Spin at 4000rpm->20min->4C 6. Remove all supernatant, leave the blue pellet 7. Wash with cold ethanol (75%) ~750ul 8. Move pellet to 1.5ml tube, vortex/mix well 9. Spin at 10,000rpm->5min->4C 10. Remove all supernatant, leave 5 min to air dry. 11. Resuspend DNA with 125 ul dH20 12. Leave in 37C incubator for 30 min. Vortex to fully dissolve DNA. 13. Pool the content of 8 tubes into one.
Step 3: Exonuclease Treatment[edit]
1. Prepare reaction (1x, 150ul):
- PCR product, 130 ul (measured)
- 10x Exo Buffer, 15 ul
- Lambdo Exonuclease (5000 U/ml), 5ul
2. Program ("Exo")
- 37C - 1 hr
- 90C - 10 min
- 4C - forever
Step 4: Purification with QuaQuick Columns[edit]
1. Add 5x PBI buffer (750 ul)
2. Mix well
3. Load equally to 2 columns
4. Spin at 14000 rpm->1min
5. Empty collection tubes
6. Add 750 ul wash buffer (PE)
7. Spin at 14000rpm->1min
8. Empty collection tubes & spin again for 2 min
9. Air dry for 5 min
10. Transfer to labeled 1.5ml tubes
11. Add 34 ul ddH20 to each center of each column
12. Wait 1 min
13. Spin at 14000rpm->1 min
NANODROP[edit]
A. 100 ng/ul 720ul total volume => 72.0 ug
Step 5: Incubation with USER Enzyme[edit]
1. Prepare reaction (1x, 74 ul)
- Probes, 60 ul
- DpnII 10x Buffer, 8 ul
- USER (1000 U/ml), 6 ul
2. Program
- 37C -> 3 hours