Dinh:Protocols/Probes Prep Nov6

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November 6, 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 (12x)
ddH20 50ul 600ul
EconoTag 2x Master Mix 1x 50ul 600ul
AP1V4IU (100uM) 0.3uM 0.3ul 3.6ul
AP2V4 (100uM) 0.3uM 0.3ul 3.6ul
SYBR Green (50x) 0.2x 0.4ul 4.8ul
Template CpG30k (20nM) 33pg 0.05ul 0.6ul
Total 101.05ul 1212.6ul

2. Program ("1 step probe preparation"):

94C - 2 min
94C - 30 s
60C - 1 min
72C - 45 s
Plate Read
Cycle to 2 for 22 more times
72C - 5 min
15C - forever
end

3. Stopped at 20 cycles.

Step 2: Ethanol Precipitation[edit]

1. In Solution Basin (55ml) Add:
NaOAc (3M), 960 ul
GlycoBlue, 32ul
Ethanol 100%, 24ml
2. Pool all reactions into Solution Basin (55ml)
3. 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. 120 ng/ul 480ul total volume => 60.0 ug

Step 5: Incubation with USER Enzyme[edit]

(Optional) Remove 1 ul for PAGE 1. Prepare reaction (1x, 72 ul)

    • Probes, 60 ul
    • DpnII 10x Buffer, 8 ul
    • USER (1000 U/ml), 6 ul

2. Program

    • 37C -> 2 hours

November 7, 2009[edit]

Step 6: Annealing of DpnII Oligo & Incubation with DpnII[edit]

(Optional) Remove 1 ul for PAGE 1. Prepare reaction (1x, 78)

    • from USER, 72 ul
    • DpnII buffer 10x, 1 ul
    • DpnII-V4 guide oligo (100uM), 5 ul

2. Program ("DpnII")

    • 95C - 10 min
    • Gradient 0.1C/s to 60C
    • 60C - 20 min
    • 37C - 4 hours
      • At 1-2 into this step, add 2ul DpnII restriction enzyme
    • 4C - forever

3. Leave overnight at 4C

November 9, 2009[edit]

Step 7: PAGE Denaturing Gel[edit]

  • Use "Probe Prep Only" gel box
  • Use PCR tubes
  • Load 2ug/2D well

1. Prepare samples:

  • 1 DpnII reaction mix: 74 ul, TBE Urea Sample Buffer (2x), 74 ul. (152ul)
  • Invitrogen 10bp ladder, 0.5ul, dH20, 4.5 ul, TBE Urea Sample Buffer (2x), 5ul.(10ul)
  • Loaded 74 ul sample+buffer mix per well
  • Loaded 5 ul ladder+buffer mix per well

2. Denature at 75C -> 8 min

3. Transfer to ice quickly.

4. Pre-run gel, 2D well, TBU gel, 200V-> 10min

5. Flush wells to remove all urea

6. Load samples quickly (hold tubes so that you are not indirectly touching the solution inside).

7. Run at 200V->30min

8. Stain with SYBR Gold (5ul in 100ml 0.5xTBE) in a clean tray.

9. Place on orbital shaker for 1-2 min.

Step 8: Purify the product from the gel[edit]

  • Switch gloves when using PCR hood

1. Prepare gel-shearing assemblies (2x) using non-stick 0.5ml and 1.6ml tubes.

2. Use G20 needle to punch a hole at the bottom of the 0.5 ml tube

3. Cut out 100-110 nt band. Divide into 2 equal pieces. Chop up each and place pieces into a gel-shearing assembly.

4. Centrifuge at 14000 rpm for 3 min to shear gel. Move any remaining pieces to bigger tube. Remove the 0.5 ml tube

5. Add 400ul clean 1x TE buffer.

6. Vortex at 37 C for 1 hour

7. In PCR hood, transfer gel mix to Nanosep columns

8. Spin at top speed 15000 rpm for 3min

9. Transfer supernatant to 1.5 ml tube (non-stick).


Step 9: Ethanol Precipitation[edit]

1. Prepare reaction:

    • Pool the content of 8 Nanosep flowthrough to 1x15ml tube. = total 6x15 ml tubes
    • NaOAc (3M), 0.1xsample vol
    • 100% ethanol, 2.5xsample vol
    • Glycoblue, 1/300th xsample vol
  • Make sure that tubes are within 0.1g of each other by adding 100% EtOH (in increments of 50ul).

2. Freeze at -70C for 20 min

3. Spin at 4000rpm->25min->4C

4. Remove supernatant

5. Wash with 500 ul 75% ethanol. Pool contents of 8 tubes into 1x1.5ml tube.

6. Spin at 8000rpm->5min->4C

7. Remove all supernatant with pipettors

8. Leave in hood to air dry ~10 min

9. Add 10ul ddH20 to each tube to resolve DNA

10. Leave at least 15 min, then leave overnight at 4C

November 11, 2009[edit]

Step 10: Quantification of DNA using Denaturing gel & a quantitative ladder[edit]

  • Combine DNA from both tubes.

1. Prepare samples:

    • Low mass DNA ladder, 3ul, dH20, 6ul, TBE urea buffer (2x), 9ul (18ul totally)
    • DNA sample, 1ul, dH20, 9 ul, TBE urea buffer (2x), 10 ul (20 ul totally)

2. Pre-run gel, 6% TBU gel, 10-12 wells, 200V->10min

3. Denature DNA + loading dye using PCR machine at 75C -> 8min

4. Transfer to ice quickly to avoid denaturation

5. Flush wells to remove urea

6. Load samples:

    • DNA: 10 ul, 5.0 ul, 2.5 ul
    • Ladder: 6 ul, 6 ul, 3 ul, 3 ul

7. Run at 200V -> 30min

8. Stain gel w/ 5ul SYBR gold in 100ml 0.5xTBE

9. Place on orbital shaker for 4 min

10. Quantify gel to calculate yield:

[[Image:|200px|thumb|left|PAGE Quantification = 12ng/ulx22ul]]