Dinh:Protocols/Probes Prep Nov2
November 2, 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.
November 5, 2009 =[edit]
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, 150ul):
- PCR product, 130 ul (measured)
- 10x Exo Buffer, 15 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. 115 ng/ul 480 ul total volume => 55.2 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 -> 2 hours
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
November 6, 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.
- Repeat QiaQuick Purification to remove salts [this step might not have been necessary.]
- Repeat DpnII Digestion.
- Repeat size selection. Ran 6 gels.
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 G20 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 6 Nanosep flowthrough to 1x15ml tube. = total 2x15 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
November 9, 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)
- 10bp ladder, 0.5ul, dH20, 4.5ul, TBE urea buffer (2x), 5 ul (10 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
- Low Mass Ladder: 6 ul, 6 ul, 3 ul, 3 ul
- 10 bp Ladder: 10ul
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: