AlanFung:LabNotes/Probe/2009-5-12

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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.

File:ZhangLab 2 2009-05-14 19hr 03min.jpg