Dinh:Protocols/Probes Prep Sep28: Difference between revisions

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** (Lanes 4-6) DNA: 9.8 ul, 5.0 ul, 2.5 ul
** (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
** (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%

Revision as of 00:03, 30 September 2009

September 23, 2009

Step 1: Real Time PCR Probe Amplification

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 (20uM), 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

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 1000rpm->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

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

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

(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

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

(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

(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

Step 7: PAGE Denaturing Gel

  • 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

  • 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

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

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

  • 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%