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