Dinh:Protocols/Probes Prep Oct27

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October 27, 2009[edit]

Step 1: Real Time PCR Probe Amplification[edit]

1. Prepare 96 Reactions (12x or 1.2ml per tube):

  • 1. ddH20, 600 ul
  • 2. EconoTag 2x Master Mix, 600 ul
  • 3. AP1V4IU (100uM), 3.6 ul
  • 4. AP2V4 (100uM), 3.6 ul
  • 5. SYBR Green (50x), 4.8 ul
  • 6. Template CpG30k (20nM), 0.6 ul

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

  • 1. 94C - 2 min
  • 2. 94C - 30 s
  • 3. 60C - 1 min
  • 4. 72C - 45 s
  • 5. Plate Read
  • 6. Cycle to 2 for 22 more times
  • 7. 72C - 5 min
  • 8. 15C - forever
  • 9. end

3. Stopped at 22 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, 139ul):

    • PCR product, 112 ul (measured)
    • 10x Exo Buffer, 14 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. 67.12 ng/ul 480 ul total volume => 32.2 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 -> 3 hours

October 28, 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

October 29, 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: 76 ul, TBE Urea Sample Buffer (2x), 76 ul. (152ul)
  • Invitrogen 10bp ladder, 0.5ul, dH20, 4.5 ul, TBE Urea Sample Buffer (2x), 5ul.(10ul)
  • Loaded 76 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 G22 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 16x15 ml tubes
    • NaOAc (3M), 0.1xsample vol
    • 100% ethanol, 2.5xsample vol
    • Glycoblue, 1/300thxsample 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

October 30, 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: