AlanFung:Protocol/Probe Preparation

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Revision as of 05:32, 1 April 2009 by >Alan6017518 (New page: Probe Preparation step 1: PCR reaction system setup Example using Dna polymerase: H2O 80.5ul 10x Stoffel buffer ...)
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Probe Preparation

step 1: PCR reaction system setup
 Example using Dna polymerase:
 H2O                                                 80.5ul       
 10x Stoffel buffer                                    10ul          
 MgCl2(25mM)                                            6ul         
 dNTP(10mM each, NEB)                                   2ul          
 primer mix                                           0.4ul         
 50x SYBG I                                           0.4ul           
 template                                             0.1ul          
 Jumpstart Taq                                        0.6ul

 Example using master mix:
 2nd Round production PCR of 27k9bp, Jan09 exome probes:
 PCR                                         x 48
 Template (10nM)                      0.2ul   9.6          
 EconoTaq 2X Master Mix                50ul  2400
 100uM AP1V41U                        0.2ul   9.6    
 100uM AP2V4                          0.2ul   9.6    
 H2O                                   50ul  2500
 SYBR green 50X                       0.4ul  19.2
   
 94C 2min -> (94C 30sec -> 58C 30sec -> 72C 30sec) x 18 -> 72C 3min -> 15C hold.
 Production PCR of probe preparation using Nicking enzyme:
                                                  x 48
 Size selected PCR amplicon           0.2ul        9.6  
 2x Taq master mix:                    50ul       2400
 50X SYBG I                           0.4ul       19.2
 100uM eMIP_CA1F                      0.2ul        9.6
 100uM eMIP_CA1R                      0.2ul        9.6
 H2O                                   50ul       2400
 94C 2min -> (94C 45sec -> 58C 30sec -> 72C 30sec) x 15 (Nicking) -> 72C 3min -> 15C hold.

 Production PCR for probe sets: 18bp, 22k, 27k probes:
 
                                                 x 48
 CES8k18bp (10nM)                     0.2ul      9.6         
 2X Taq Master Mix                     50ul      2400
 100uM AP1V6                          0.2ul      9.6    
 100uM AP2V6                          0.2ul      9.6    
 H2O                                   50ul      2400 
 50X SYBG I                           0.4ul      19.2
 94C 2min -> (94C 30sec -> 60C 30sec -> 72C 30sec) x 19 -> 72C 3min -> 15C hold. 
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 100 uL dH2O.


Step 3: Purify the reaction with Qiaquick columns
1. Add 5X the amount of PB buffer into each tube and load the mixture to the 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 on bench top, wait for 2 min
8. Add 50 uL ddH2O to each column, wait for 1 min, spin at top speed for 1 min.
   NOTE: Add 100ul of ddH2O to probe using nicking enzyme
9. measure the concentration with Nanodrop.

Step 4: Adapter Removal
For Jan09 exome Sets #1-4:
1. To each tube, add 10ul Dpn II buffer, 36ul H2O, 4ul Dpn II(50unit/ul).
2. Incubate at 37C for 1hr and 75C 10min. 
3. USER enzyme digestion: add 10ul USER enzyme, and incubate at 37C for 4hr
4. Purified the products with Qiaquick columns, eluted with 60ul ddH2O. 
For Jan09 exome Sets #5:
1. Add 6 uL 10X Exo buffer and 8 uL Lambdo Exonuclease (5U/ul).
2. Incubate at 37C for 2 hours followed by 90C for 10 min
3. perform one round of Qiaquick column purification and elute in 50ul of H2O each
3. To each tube, add 10ul Dpn II buffer, 36ul H2O, 4ul Dpn II(50unit/ul).
4. Incubate at 37C for 1hr and 75C 10min. 
5. USER enzyme digestion: add 10ul USER enzyme, and incubate at 37C for 4hr
6. Purified the products with Qiaquick columns, eluted with 60ul ddH2O. 
For Probe using Nicking Enzyme digestion:

purified PCR product:         50ul
10x NEBuffer2:                10ul
H2O                           35ul
Nt.AlwI (10U/ul)               5ul

1. Incubate at 37C for 3 hours
2. perform one round of Qiaquick column purification and elute in 100ul of ddH2O
3. add 2ul Nb.BsrDI and incubate at 65C for 3hr
4. perform one round of Qiaquick column purification and elute in 60ul of ddH2O
For 18bp, 27k9bp, 27k, and 22k sets:
1. Add 6 uL 10X Exo buffer and 8 uL Lambdo Exonuclease (5U/ul).
2. Incubate at 37C for 2 hours followed by 90C for 10 min
3. perform one round of Qiaquick column purification and elute in 50ul of H2O each 
4. Add 10ul 10X DpnII buffer, 5ul 100uM of guide oligo:
   (DpnIIV4 for 27k9bp probe set, and RE-DpnII-n2s for 18bp probe set and 27k set, RE_RE_DpnII_n2s for 22k set)
5. 94C 5min -> 60C 5min -> 0.1C/sec to 37C -> 37C 1min -> add 4ul DpnII restriction enzyme -> 37C 2hr
6. Add 4ul of USER enzyme -> 37C 4hr
7. Purified the products with Qiaquick columns, eluted with 60ul ddH2O. 


Step 5: 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 8 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 6: 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. Use 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 7: 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.
Step 8: Quantification of DNA using denaturing gel and a quantitative ladder
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 15 min, quickly transfer to ice, 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.