AlanFung:LabNotes/Probe/2009-5-12
Jump to navigation
Jump to search
Purpose[edit]
- To improve my prob preping skills
- Be extremely careful with contamination, clean every tools and equipments before use
- Following kun's protocol exactly the way he did it on 04/16/09
- No SYBRgreen added
- No column purification after ethanol precipitation
- Dilute 24 wells 1 to 6 for column purification
Step 1: PCR reaction system setup[edit]
- Thaw Econo Taq on ice
- Prepare 6 strips and 4x 2ml tubes on cooling rack
- Add in Content in table to tubes accordingly
Content | Volume | X48 | 2 mL Tube | 2 mL Tube | |
RNAse free H2O | 50uL | 2400uL | 600uL | 600uL | |
AP1V6 AP2V6 Mix (100uM) | 0.2uL | 9.6uL | 2.4uL | 2.4uL | |
AP1V6 AP2V6 Mix (100uM) | 0.2uL | 9.6uL | 2.4uL | 2.4uL | |
Jan_09_CPG_D40809 (10uM) | 0.2uL | 9.6uL | 2.4uL | 2.4uL | |
Econo Taq | 54.17uL | 2600uL | 650uL | 650uL |
- Mix content following the order from top to bottom in 4x 2 mL tube vortex and spin down
- Aliquot 104uL using P200 pipette to strip tubes on cooling rack
- Seal strips with strip caps
- Place into Real Time PCR
- Perform
94C 2M > (94C 30S > 58C 30S > 72C 30S) X16 > 72C 3M > 15C Hold
Step 2: Probe purification with ethanol precipitation[edit]
- Add in required volume of 100% ethanol, 3M NaOAC and Glycoblue into tray
Content | Volume Needed | Volume |
100% Ethanol | 2.5X Sample Volume | 12572uL |
3M NaOAC | 0.1X Sample Volume | 502.88uL |
Glycoblue | 1/300X Sample Volume | 16.76uL |
Sample | Sample Volume | 5028.8uL |
Total Volume | 18120.44uL |
- Pool all 48 wells PCR product into tray using multichannel pipette(P300)
- Mix all content by repeat pipetting using 10mL Pipette
- Aliquot to 2 x 15mL tubes
- Store in -80C for more than 20 mins
- Turn on Centrifuge fix temperature at 4C
- Centrifuge frozen 15mL tubes at 3000rpm for 20m at 4C
- Remove all liquid CAUTION: DO NOT DISCARD BLUE PELLET AT THE BOTTOM OF THE TUBE
- Add in 350uL cold 75% Ethanol transfer pellet to 1.5mL tube
- Addin another 350uL 75% ethanol wash the 15ml tube and transfer to 1.5mL tube
- Centrifuge at 10,000rpm for 5min at 4C
- Remove all liquid, air dry sample for 5min or until samples dried out
- Resuspend the DNA with 100uL RNAse free H2O in 2 x 1.5mL tubes
- take out 20uL from tube A perform qiaquick and nanodrop
*OD 260/280:1.95 46ng/uL*50uL
Step 4: Adapter Removal[edit]
Exonuclease treatment In a PCR tube
- Combine the 50uL qiaqick purified amplicons with 80uL sample (tubeA)
- To tube A add 4uL Lambda Exo, 13uL 10X Lambda Exo buffer
- To tube B add 4uL Lambda Exo, 11uL 10X Lambda Exo buffer
- Incubate at37C 30min>4C hold Saved as program Alan>EXOAR
Purify the reaction with Quaquick columns
- Aliquot both tube A and B in to 12 aliquots (~24uL per pcr tube)
- Add 6X sample volume of PB buffer a 1.5mL tube, mix well
- Load mixture onto column
- Centrifuge at 14,000rpm for 1min
- Discard flow through and add 750uL PE buffer to each column
- Repeat Centrifuge
- Discard flow through
- Repeat centrifuge
- Transfer columns to clean 1.5mL tubes
- Airdry the column/tube assemblies
- For two tubes in A and two tubes in B add in 50uL EB buffer to each column let stand for 5 min
- For the remaining tubes add in 30 uL EB buffer to each column let stand for 5 mins
- Repeat centrifuge
- Measure concentration with Nanodrop
*A(50uL EB buffer) 260/280 OD:1.82 15.9ng/uL *A(50uL EB buffer) 260/280 OD:1.82 11.9ng/uL *A(30uL EB buffer) 260/280 OD:1.72 30.4ng/uL
*B(50uL EB buffer) 260/280 OD:1.88 14.7ng/uL *B(30uL EB buffer) 260/280 OD:1.82 23.1.4ng/uL
- Pool and aliquot 110uL sample to 4 pcr tube
- Add in 2.5uL USER (1K/mL) enzyme into each tube incubate for 30m at 37C
Annealing of DpnII oligo and incubation with DpnII.
- Add 11uL of 10X DpnII buffer and 2.5uL RE-DpnIIs guide oligo (100uM) to each tube
- Perform 94C 2m > 0.1C/sec to 37C > add 2.5ul of DpnII restriction enzyme to each tube -> 37C 2 hours > 75C 20 min > 37 4 hours > 4C hold
Step 5: Page Denaturing Gel[edit]
- Prepare a 2D well denaturing gel by scraping off the gel residue outside and peel of the sticker at the back of gel
- Load 2 gels into the gel running module
- Add in fresh 0.5X TBE buffer to the inside core full the outer core up to the metal line
- Pre-run the gel for 30 min at 200V
- Dilute sample to no more than 40ng/uL
- Dilute TBE-Urea sample buffer (2X) to 1X in each tube.
For 2 gels; Mix 0.5 uL Invitrogen 10bp ladder with 4.5 uL dH2O, 5 uL 2x TBE-Urea sample buffer For 4 gels; Mix 0.5 uL Invitrogen 10bp ladder with 9.5 uL ddH2O, 10uL 2X TBE-Urea sample buffer
- Denature samples and ladder at 75C for 7 min, then quickly transfer to cooling rack, wait for 5 mins
- Flush the wells very well with P1000 & P200 pipette to remove all urea.
NOTE: DO NOT TOUCH THE TUBE WITH YOUR HANDS IN THE FOLLOWING STEP, GRAB THE CAP
- Load the samples into the wells, run at 200V for 30 min.
- Stain gel with 4uL SYBR Gold in ~100 mL of TBE in clean tray.
- Place tray on an orbital shaker for ~5 min.
Step 6: Purify the product from the gel[edit]
- Prepare two gel shearing assemblies by placing a 0.5 mL tube within another 1.5 mL Non-Stick tube. Use G20 needle to punch a small hole at the bottom of the 0.5 mL tube. Use two assemblies for each PAGE gel.
- Cut out the correct band (85-92 nt in this case), chop the band into small slices, split into two half and transfer each half to one shearing assembly.
- Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube.
- Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 37C incubator, shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight.
- Spin down all tubes at 13,000rpm for 2min
- In the PCR hood, transfer the liquid layer of the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm), and then transfer supernatant to 1.5mL tube.
File:ZhangLab 2 2009-05-14 11hr 58min.jpg
Step 7: Precipitation[edit]
Turn on centrifuge let it cool down to 4C
- Add 0.1x total volume of 3M sodium acetate and 2.5x total volume 100% ethanol.
- Add 1uL Glycoblue vortex and spin down so that one can precipitate better and see the DNA.
- Freeze in -80C for 20 min, spin at 10,000 rpm for 20 min at 4C, discard supernatant with pipette.
- Add 500 uL of 75% ethanol to wash DNA, spin at 10,000 rpm for 5 min at 4C, discard supernatant and aspirate with (P1000 pipette) let dry for 5 mins spindown ad aspirate with P200 pipette.
- Let dry in hood ~ 10 min.
- Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
Store at 4C if needed, spin down before proceeding
Step 8: Quantification of DNA using denaturing gel and a quantitative ladder[edit]
Combine all tubes into a 1.5mL tube
1. Add 4uL of 5ng/uL 90-mer, 4ul of H2O and 8 uL of 2x TBE-Urea buffer 2. Dilute sample 10x (1uL sample + 9 uL of water) so total is 10uL of sample, and add 10 uL of 2x TBE buffer 3. Denature the DNA with loading dye on thermalcycler 75C for 7 min, quickly transfer to cooling rack, wait for 1 min. 4. Use 6% 10 well TBU gel, add 0.5x TBE buffer, flush the wells very well to remove all urea. 5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min. Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL. 6. Stain the gel with 5 uL SYBR gold in ~100 mL of TBE in clean tray. Place the tray on an orbital shaker for 5 min. Then quantify the gel to calculate the % yield.