Dinh:Protocols/Probes Prep Oct6
October 6, 2009[edit]
Step 1: Real Time PCR Probe Amplification[edit]
1. Prepare 96x3 Reactions (50 ml tube):
- 1. ddH20, 14.112 ml
- 2. EconoTag 2x Master Mix, 14.400 ml (11 full tubes + 650 ml)
- 3. AP1V4IU (100uM), 86.4 ul
- 4. AP2V4 (100uM), 86.4 ul
- 5. SYBR Green (50x), 115.2 ul
- 6. Template CpG30k (20nM), 14.4 ul
--> Use 200 ul flat strip tubes
2. Program:
- 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 in a plate 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 30 min to air dry.
11. Resuspend DNA with 125 ul dH20
12. Incubate at 37C for 30 min.
13. Resuspend by pipetting. Transfer to 200 ul PCR tubes/strip.
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 QiaQuick 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 15 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
15. Combine 2x30ul into 1 PCR tube.
16. Leave in 4C overnight.
October 7, 2009[edit]
NANODROP[edit]
A. 115.8 ng/ul x 4 x 48ul = 22.4 ug B. 288.8 ng/ul x 4 x 48ul = 55.5 ug C. 268.0 ng/ul x 4 x 48ul = 51.5 ug
Step 5: Incubation with USER Enzyme[edit]
1. Prepare reaction (1x) Add:
- DpnII 10x Buffer, 8 ul
- USER (1000 U/ml), 6 ul
2. Program
- 37C -> 3 hours, stopped temporarily at 2.5 hr to remove 1 ul from each tube for PAGE qualification.
3. Ran gel to check digestion.
Step 6: Annealing of DpnII Oligo & Incubation with DpnII[edit]
1. Prepare reaction (1x) Add:
- 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
October 8, 2009 - October 14, 2009[edit]
- October 8-9 - Set Y (1 plate)
- October 13-14 - Sets P & B (2 plates)
Step 7: PAGE Denaturing Gel[edit]
- Use "Probe Prep Only" gel box
- Use PCR tubes
1. Prepare samples:
- Probes, 70 ul, TBE Urea Sample Buffer (2x), 70 ul. (140ul)
- 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 (3ul in 100ml 0.5xTBE) in a clean tray.
9. Place on orbital shaker for 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 15 ml tube (sterile, RNA-ase, DNA-ase free). (For Set Y, supernatant were transfered to individual non-stick tubes)
Step 9: Ethanol Precipitation[edit]
1. Prepare reaction:
Set Y: (for individual 1.5ml tubes) vol = 400 ul each
- NaOAc (3M), 40 ul
- 100% ethanol, 1000ul
- Glycoblue, 1 ul
Set P: (for 15ml tube) vol = 3.50 ml
- NaOAc (3M), 350 ul
- 100% ethanol, 8.25 ml
- Glycoblue, 8 ul
Set B: (for 15ml tube) vol = 4.00 ml
- NaOAc (3M), 400 ul
- 100% ethanol, 10.00 ml
- Glycoblue, 8 ul
2. Freeze at -70C for 20 min
3. Spin at 13700rpm->15min->4C / for 1.5 ml tubes, 3700rpm->30min->4C for 15 ml tubes
4. Remove supernatant
5. Wash with 750 ul 75% ethanol, transfer pellets to one 1.5 ml tube (non-stick)
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
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:
Set Y:
- (Lanes 2-5) Ladder: 5.6 ul, 5.6 ul, 3.0 ul, 3.0 ul
- (Lanes 6-9) DNA: 10.0 ul, 5.0 ul, 2.5 ul, 2.5 ul
Set P:
- (Lanes 2-5) DNA: 10.0 ul, 5.0 ul, 2.5 ul, 2.5 ul
- (Lanes 6-9) Ladder: 6 ul, 6 ul, 3.5 ul, 3.5 ul
Set B:
- (Lanes 2-4) DNA: 10.0 ul, 5.0 ul, 2.5 ul
- (Lanes 5-8) Ladder: 3 ul, 3 ul, 6 ul, 6 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.