Alice:LabNotes/2008-5-12

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cosmic 3760 probe preparation

Step 1: Real time PCR for cosmic probe

 reaction system                                               
 H2O                                     84.2ul        1347.2ul     
 10xBuffer                                 10ul           160ul         
 dNTP(10mM)                                 4ul            64ul         
 Primer Mix (100uM): Ap1V4IU +Ap2V4         1ul            16ul      
 SYBR  (50x)                              0.5ul             8ul 
 Jumpstart Taq                            0.2ul           3.2ul
 template(cosmic3760)                     0.1ul           1.6ul 
 Total                                    100ul          1600ul

Primer sequence: forward primer Ap1V4IU and reverse primer Ap2V4

Reaction program:

94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 22 cycles -> 72C 5min -> 15C hold.

Note: 32 tubes were prepared instead of 16.

Step 2: Probe purification with 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 15 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 250 uL dH2O. Transfer the liquid to two 0.2ml tube.

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 4 hours 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 750 uL of PBI buffer, mix well, load 450 uL x4 to each column 
 2. Spin at top speed (14000 rpm) for 1 min
 3. Empty the collection tube, add 750 uL wash buffer (PE buffer) to the column
 4. Spin at top speed for 1 min
 5. Empty the collection tube, spin at top speed for 1 min
 6. Carefully transfer the columns to clean 1.6 mL tubes
 7. Place the column/tube assemblies to 37C incubator, incubate for 5 min
*8. Add 65 uL ddH2O to each column, wait for 1 min, spin at top speed for 1 min.
 measure the concentration with Nanodrop.

Result: tube 1: 2.3 ng/ul in 65 ul of sample, total yield is 149.5 ng. tube 2: 2.8 ng/ul in 65 ul of sample, total yield is 182 ng.

Step 5: Incubation with USER enzyme:

*1. Add 8uL of 10X DpnII reaction buffer and 10 uL of USER (1K/ml)enzyme and last 8 hour on 37C. 
 Take 4ul out to run gel later.

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

*1. Add 8 uL of 100 uM DpnII-V4 guide oligo.
 2. Denature at 95C in PCR machine for 10 min.
 3. Turn off the PCR program and let samples cool off in the block for ~15 min (the temperature cools from 95C to around 40C).
 4. Add 10 uL of DpnII restriction enzyme (10K/ml) to reaction, incubate for about 4 hours at 37C, take 4ul out to run the gel.


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 the DNA with loading dye on thermalcycler 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.

File:ZhangLab 2 2008-05-17 17hr 58min.jpeg

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 8uL of 2ng/uL 90-mer 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-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 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 2008-05-19 14hr 14min.jpeg