AlanFung:LabNotes/CPG 30K/2009-5-20: Difference between revisions

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>Alan6017518
 
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==Procedures==
==Procedures==
==Step 1: PCR reaction system setup==
==Step 1: PCR reaction system setup==
*Thaw Econo Taq on ice 8tubes/plate
*Thaw Econo Taq on ice 8 tubes/plate
*Prepare strips and tubes on cooling rack
*Prepare strips and tubes on cooling rack
*Add in Content in table to tubes accordinly
*Add in Content in table to tubes accordinly
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|-
|-
|CPG_30K (20uM)||align="right"|0.025uL||align="right"|0.6uL||align="right"|1.2uL||align="right"|1.8uL||align="right"|2.4uL
|CPG_30K (20nM)||align="right"|0.1uL||align="right"|2.4uL||align="right"|4.8uL||align="right"|7.2uL||align="right"|9.6uL
|-
|-
|Econo Taq||align="right"|50uL||align="right"|1250uL||align="right"|2400uL||align="right"|3600uL||align="right"|4800uL
|Econo Taq||align="right"|50uL||align="right"|1250uL||align="right"|2400uL||align="right"|3600uL||align="right"|4800uL
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*Mix all content in tray in the order of water>SYBR Green>Primer >template>econotaq  
*Mix all content in tray in the order of water>SYBR Green>Primer >template>econotaq  
*Mix well with P1000
*Mix well with P1000
*Aliquot 100uL using multi-channel pipette to strip tubes on cooling rack  
*Aliquot 100.9 uL using multi-channel pipette to strip tubes on cooling rack  
*Seal strips with strip caps
*Seal strips with strip caps
*Place into Real Time PCR
*Place into Real Time PCR
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*Spin down all tubes at 13,000rpm for 2min  
*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.
*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.
[[Image:ZhangLab_2 2009-05-24 20hr 32min.jpg]]


==Step 7: Precipitation==
==Step 7: Precipitation==

Latest revision as of 03:53, 25 May 2009

Probe Preparation[edit]

Objective[edit]

  • Synthesis of CPG_30K Probe Set (Whole Plate)

Samples & Materials[edit]

Overview[edit]

  • PCR reaction system setup
  • Probe purification with ethanol precipitation
  • Purify the reaction with Qiaquick columns
  • Adapter Removal
  • Page Denaturing Gel
  • Purify the product from the gel
  • Precipitation
  • Quantification of DNA using denaturing gel and a quantitative ladder

Procedures[edit]

Step 1: PCR reaction system setup[edit]

  • Thaw Econo Taq on ice 8 tubes/plate
  • Prepare strips and tubes on cooling rack
  • Add in Content in table to tubes accordinly
Content Volume X24 X48 X72 X96
CPG_30K (20nM) 0.1uL 2.4uL 4.8uL 7.2uL 9.6uL
Econo Taq 50uL 1250uL 2400uL 3600uL 4800uL
AP1V41U (100uM) 0.3uL 7.2uL 14.4uL 21.6uL 28.8uL
AP2V4(100uM) 0.3uL 7.2uL 14.4uL 21.6uL 28.8uL
RNAse free H2O 50uL 1200uL 2400uL 3600uL 4800uL
50X SYBG I 0.4uL 9.6uL 19.2uL 28.8uL 38.4uL
Total Volume 101uL 2424uL 4848uL 7272uL 9686.4uL
  • Mix all content in tray in the order of water>SYBR Green>Primer >template>econotaq
  • Mix well with P1000
  • Aliquot 100.9 uL using multi-channel 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>Plate Read) X20-22 > 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 24216uL
3M NaOAC 0.1X Sample Volume 968.64uL
Glycoblue 1/300X Sample Volume 32.288uL
Sample Sample Volume 9686.4uL
Total Volume 34903.328uL
  • Mix all content by repeat pipetting using multichannel pipette
  • Pool all 24 wells PCR product into tray using multichannel pipette(P300)
  • Mix all content by repeat pipetting using multichannel pipette
  • Aliquot about 17451uL to 2 x 15mL tube
  • Store in -80C for more than 20 mins or forever
  • Turn on Centrifuge fix temperature at 4C, place a tube of 75% ethanol into 4C fridge
  • 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 750uL cold 75% Ethanol, mix by repeat pipetting and transfer to a 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


Step 4: Adapter Removal[edit]

Exonuclease treatment

*8ul Lambda Exo/48wells
  • Add in 8uL Lambda Exonuclease (5K/mL) to each tube for sample concentration around 100ng/uL NOTE: If sample too concentrated, dilute sample to about 100ng/uL per tube


  • Add in (1/10 of total volume) 6uL 10X Exo buffer
  • Incubate at37C 4h>90C 10m>4C hold Saved as program Alan>EXOAR


Purify the reaction with 12 Quaquick columns (1 strip PCR tubes/column)

  • Add 5X sample volume of PB buffer into each 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
  • Place the column/tube assemblies on bench top wait for 2min
  • Add in 50uL EB buffer to each column (For nicking enzyme probes add in 100ul of ddH2O) let stand for 5 min
  • Repeat centrifuge
  • Measure concentration with Nanodrop
*5 uL USER/24wells
  • Combine 12 tubes to 4 tubes
  • Add 40ul ddH2O, 5 ul USER to each tube
  • Incubate at 37C for 1h
*5uL DpnII-V4 guide oligo/24wells
*5uL DpnII/24wells
  • Add 10ul 10x DpnII buffer, 5 ul DpnII-V4 guide oligo,

94C 2min -> 37C 3min -> add 5 ul DpnII to each tube -> 37C 6h -> 75C 20min -> 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 (~110 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 2009-05-24 20hr 32min.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.