Dinh:Protocols/Probes Prep Sep28: Difference between revisions

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7. Spin at 14000rpm->1min
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.

Revision as of 23:43, 29 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.