Jie:LabNotes/Protocol/probe library construction

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Revision as of 19:49, 28 September 2009 by >Sam Chiang
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probe preparation[edit]

Step 1: Real time PCR

 reaction system                                                  x12       x8        
 H2O                                                70.5ul      846ul    6768ul  
 10x hotstart Taq buffer                              10ul      120ul     960ul
 MgCl2(25mM)                                           6ul       72ul     576ul
 dNTP(10mM;NBE)                                        2ul       24ul     192ul
 Ap1V4IU(100uM)                                      0.2ul      2.4ul    19.2ul
 Ap2V4(100uM)                                        0.2ul      2.4ul    19.2ul
 50x SYBG I                                          0.4ul      4.8ul    38.4ul
 template(20nM)                                      0.1ul      1.2ul     9.6ul
 hotstart Taq                                        0.6ul      7.2ul    57.6ul
 Total                                               100ul     1200ul    9600ul
 Reaction program: 94c 2min -> (94c 30sec -> 60C 1.5min -> 72C 1min)x 20~22 cycles -> 72C 5min -> 15C hold.

step 2: ethanol precipitation

 1. pool the 24 reactions into one 16 mL tube, add 240 uL 3M NaOAC, 8 uL GlycoBlue, 
    6 mL 100% Ethanol.
 2. Store at -70C for more than 20 min.
 3. Spin at 4000rpm for 25 min at 4C.
 4. Remove all liqid, you should see a blue pallette at the bottom of the tube.
 5. Add 750 uL cold 75% Ethanol, piette well and transfer to a 1.6 mL tube.
 6. Spin at 10000 rpm for 5 min at 4C.
 7. Remove all liquid, let the pallette dry for 5 min, resuspend the DNA with 125 uL dH2O. 

Step 3: Exonuclease treatment

 1. Add 15 uL 10X Exo buffer and 8 uL Lambdo Exonuclease (5K/ml) for total volume of 150uL(each tube).
 2. Incubate at 37C for 1 hour followed by 90C for 10 min, and leave it at 4C forever.

Step 4: Purify the reaction with Quaquick columns

 1. for each tube, add 5x PBI buffer, mix well, load to each column 
 2. Spin at top speed (14000 rpm) for 1 min. Empty the collection tube, add 750 uL wash buffer (PE buffer) to the column.Spin at top speed for 1 min
 3. Empty the collection tube, spin at top speed for 1 min, transfer the columns to clean 1.6 mL tubes
 4. Place the column/tube assemblies to roomtemprature for several mins.
 5. Add 45 uL ddH2O to each column, wait for 1 min, spin at top speed for 1 min.
 measure the concentration with Nanodrop.
 

Step 5: Incubation with USER enzyme:

 Add 6ul DpNII 10x buffer and 8 uL of USER (1K/ml)enzyme and last 2 hour on 37C. 
 

Step 6: Annealing of DpnII oligo and incubation with DpnII.

 1. Add 1uL of 10X DpnII reaction buffer and 10 uL of 100 uM DpnII-V4 guide oligo. Denature at 95C in PCR machine for 10 min.
 2. cool 0.1C/sec to 60C and hold 60C for 20mins -> 37C 1min -> Add 2 uL of DpnII restriction enzyme (50K/ml) to reaction, incubate for about 4 hours at 37C.
 

Step 7: Page Denaturing Gel

 1. Add TBE-Urea sample buffer (2x) in each tube.
 2. Mix 0.5 uL Invitrogen 10bp ladder with 4.5 uL dH2O, 5 uL 2x TBE-Urea sample buffer.
 3. Denature at 75C for 15 min, then quickly transfer to ice, wait for 1 min.
 4. Use a 2D well denaturing gel, add 0.5x TBE buffer. Flush the wells very well to remove all urea. 
 6. Pre-run the gel for 10 min at 200V, load the samples into the wells, run at 200V for 30 min.
 7. Stain gel with 5uL SYBR Gold in ~100 mL of TBE in clean tray. Place tray on an orbital shaker for ~5 min.

Step 8: Purify the product from the gel

 1. 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. You need two assemblies for each PAGE gel. 2.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. 3. Centrifuge at top speed > 12000 rpm for 2 min to sheer the gel, remove the 0.5 mL tube. 4. Add 400 uL clean 1x TE buffer in the 1.5 mL tube. Tape the tubes on a vortexer, place the vortexer in 50C incubator,
shake the tubes at low speed for 1 hour. Alternatively, can keep the tubes in 4C fridge overnight. 5. In the PCR hood, transfer the gel mix to Nanosep columns, spin for 3 min at top speed (15,000 rpm),
and then transfer supernatant to 1.5mL tube.


Step 9: Precipitation

 1. Add 0.1x of 3M sodium acetate and 2.5x 100% ethanol.
 2. Add 1uL Glycoblue so that one can precipitate better and see the DNA.
 3. Freeze in -70C for 20 min, spin at 14,000 rpm for 15 min at 4C, discard supernatant with pipette.
 4. Add 500 uL of 75% ethanol to wash DNA, spin at 8000 rpm for 5 min at 4C, discard supernatant with 
first large tip and then small tip to remove as much liquid as possible. 5. Let dry in hood ~ 10 min. 6. Add 20 uL of water(totally) to the tube to resolve the DNA in, leave at least 15 min.
  • I transfer 500ul 75% ethanol and DNA into another tube and another to accumulate the probe.

Step 10: Quantification of DNA using denaturing gel and a quantitative ladder

 1. Add 3uL of low mess ladder and 6ul H2O and 9 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-Urea buffer.
 3. Denature the DNA with loading dye on thermalcycler 75C for 15 min, then quickly transfer to ice, wait for 1 min.

Note: Transfer to ice to keep the samples from further denaturation.

 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 3, 3, 6, 6 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.