Dinh:Protocols/Probes Prep Sep28

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September 23, 2009[edit]

Step 1: Real Time PCR Probe Amplification[edit]

1. Prepare 24 Reactions (12x or 1.2ml per tube):

  • 1. ddH20, 588 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 23 cycles.

Step 2: Ethanol Precipitation[edit]

1. Pool all reactions into Solution Basin (55ml)

2. Add:

    • NaOAc (3M), 260 ul
    • GlycoBlue, 8ul
    • Ethanol 100%, 6ml

3. Transfer to 15 ml tube. Store at -70C for more than 20 min

4. Turn on centrifuge (might need to switch the "swirlers")

5. Spin at 3700rpm->20min->4C

6. Remove all supernatant, leave the blue pellet

7. Wash with cold ethanol (75%) ~750ul

8. Move pellet to 1.6ml tube, vortex/mix well

9. Spin at 10,000rpm->5min->4C

10. Remove all supernatan, leave 5 min to air dry.

11. Resuspend DNA with 125 ul dH20

12. Leave in 4C overnight.

September 24, 2009[edit]

13. Make sure the DNA is homogenized in solution by leaving in 37C incubator for 25 min.

14. Transfer DNA to PCR tubes

Step 3: Exonuclease Treatment[edit]

1. Prepare reaction (1x, 150ul):

    • PCR product, 116 ul (measured)
    • 10x Exo Buffer, 15 ul
    • Lambdo Exonuclease (5000 U/ml), 5ul
    • ddH20, 14 ul

2. Program ("Exo")

    • 37C - 1 hr
    • 90C - 10 min
    • 4C - forever

Step 4: Purification with QuaQuick Columns[edit]

(Optional) 1ul sample with 4ul dH20 for PAGE 1. Add 5x PBI buffer (790 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 1.6ml tubes

11. Add 30 ul ddH20 to each center of each column

12. Wait 1 min

13. Spin at 14000rpm->1 min

14. Pipette remaining dH20 through the center and spin again at 14000rpm->1min

NANODROP[edit]

A. 101.7 ng/ul x 24ul B. 91.7 ng/ul x 24ul

48 ul total volume => 4.642 ug

Step 5: Incubation with USER Enzyme[edit]

(Optional) Remove 1 ul for PAGE 1. Prepare reaction (1x, 62 ul ! varies)

    • Probes, 48 ul
    • DpnII 10x Buffer, 8 ul
    • USER (1000 U/ml), 6 ul

2. Program

    • 37C -> 1-2 hours

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

(Optional) Remove 1 ul for PAGE 1. Prepare reaction (1x, 68 ul ! varies)

    • from USER, 62 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

September 25, 2009[edit]

Step 7: PAGE Denaturing Gel[edit]

  • Use "Probe Prep Only" gel box
  • Use PCR tubes

1. Prepare samples:

  • Probes, 67 ul, TBE Urea Sample Buffer (2x), 67 ul. (134ul)
  • Invitrogen 10bp ladder, 0.5ul, dH20, 4.5 ul, TBE Urea Sample Buffer (2x), 5ul.(10ul)

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 85-9 nt band. Divide into 2 equal pieces. Chop up each and place pieces into a gel-shearing assembly.

4. Centrifuge at top-speed > 12000 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:

    • NaOAc (3M), 40 ul
    • 100% ethanol, 1000ul
    • Glycoblue, 1 ul

2. Freeze at -70C for 20 min

3. Spin at 13700rpm->15min->4C

4. Remove supernatant

5. Wash with 500 ul 75% ethanol

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

September 28, 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:

    • Lane 1: Probe before Quaquick
    • Lane 2: After Quaquick
    • Lane 3: After USER enzyme
    • (Lanes 4-6) DNA: 9.8 ul, 5.0 ul, 2.5 ul
    • (Lanes 7-10) Ladder: 5.8 ul, 5.8 ul, 2.8 ul, 2.0 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: 1.91/4.64 ug = 41%

File:ZhangLab 2 2009-09-28 Dinh Diep gel quantification.jpg