Dinh:Protocols/Probes Prep Sep28
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%