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Experiment: Cosmic 3760 probe preparation
<b>Experiment 1: Cosmic 3760 probe preparation</b>
<br>
<br>
Step 1: Real time PCR for cosmic probe
Step 1: Real time PCR for cosmic probe
 
  Reaction System                                        X8          X12
  reaction system                                             
  H2O                                    84.8ul     678.4 ul    1017.6 ul      
  H2O                                    84.8ul       1356.8ul      
  10xBuffer                                10ul        80 ul      120 ul
  10xBuffer                                10ul           160ul          
SYBR  (50x)                              0.5ul          4 ul        6 ul
  dNTP(10mM)                                4ul           64ul          
*Mix the tube very well using vortex at this point to avoid locally concentrated
  Primer Mix (100uM): Ap1V4IU +Ap2V4      0.4ul           6.4ul
SYBR*       
  SYBR  (50x)                             0.5ul            8ul
  dNTP(10mM)                                4ul         32 ul        48 ul          
  Jumpstart Taq                            0.2ul           3.2ul
  Primer Mix (100uM): Ap1V4IU +Ap2V4      0.4ul       3.2 ul      4.8 ul
template(cosmic3760)                    0.1ul          1.6ul
  template(cosmic3760)                     0.1ul        0.8 ul      1.2 ul
Total                                    100ul         1600ul
  Jumpstart Taq                            0.2ul       1.6 ul      2.4 ul
  Total                                    100ul       800 ul      1200 ul


Primer sequence: forward primer Ap1V4IU and reverse primer Ap2V4
Primer sequence: forward primer Ap1V4IU and reverse primer Ap2V4
Line 17: Line 18:
Reaction program:
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.
94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 22 cycles -> 72C 5min -> 15C hold.<br>


Note: 8 tubes were prepared and ran first to test if the reaction system is working properly.


Step 2: Probe purification with ethanol precipitation
  Result: The PCR reaction did not work as the curve never shot up. I am suspecting
  either the Taq enzyme or the 10x buffer is not working, so I will do a comparison to
  test the hypothesis. Since I ran a PCR reaction with Platium Taq and buffer few weeks
  ago, and it worked fine. So it will be used as reference.


1. pool the 16 reactions into one 10 mL tube, add 160 uL 3M NaOAC, 5 uL GlycoBlue,
<b>Experiment 2: Testing the effectiveness of Jumpstart Taq and buffer</b>
    3.2 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 top speed for 5 min at 4C.
7. Remove all liquid, let the pallette dry for 5 min, resuspend the DNA with 125 uL dH2O. Transfer the liquid to two 0.2ml tube.


Step 3: Exonuclease treatment
Reaction Systems                                             


1. Add 15 uL 10X Exo buffer and 8 uL Lambdo Exonuclease (5K/ml) for total volume of 150uL(each tube).
  reaction system 1:                                             
  2. Incubate at 37C for 4 hours followed by 90C for 10 min, and leave it at 4C forever.
  H2O                                    85.1ul         
  10xBuffer                                10ul             
  dNTP(10mM)                                 4ul                 
  Primer Mix (100uM): Ap1V4IU +Ap2V4      0.4ul         
  SYBR (50x)                              0.5ul           
  Platium Taq                              0.5ul
  template(cosmic3760)                    0.1ul         
  Total                                    100ul


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
  reaction system 2:                                                
2. Spin at top speed (14000 rpm) for 1 min
  H2O                                                  78ul     
3. Empty the collection tube, add 750 uL wash buffer (PE buffer) to the column
  10x Platium Taq buffer                               10ul
4. Spin at top speed for 1 min
  Jumpstart Taq polymerase                            0.2ul
5. Empty the collection tube, spin at top speed for 1 min
  MgCl2(25mM)                                           6ul       
6. Carefully transfer the columns to clean 1.6 mL tubes
  dNTP(10mM)                                           4ul              
7. Place the column/tube assemblies to 37C incubator, incubate for 5 min
  primer mix (100uM)                                 0.4ul       
*8. Add 65 uL ddH2O to each column, wait for 1 min, spin at top speed for 1 min.
  50x SYBG I                                          0.4ul       
measure the concentration with Nanodrop.
  template(cosmic 3760)                              0.1ul     
 
  Total                                              100ul
 
<br>
Step 5: Incubation with USER enzyme:
<b>Result:</b>
 
 
*1. Add 8uL of 10X DpnII reaction buffer and 10 uL of USER (1K/ml)enzyme and last 8 hour on 37C.
  [[Image:6-24-08 cosmic PCR.jpg]]
Take 4ul out to run gel later.
<br>
 
The PCR for both tubes didn't work, thus suggested that the Jumpstart Taq and enzyme were not working properly. However, a control group with platium Taq and buffer was not included, so we couldn't conclude that only the Jumpstart Taq and buffer were causing the problem. Therefore another experiment with control group will be repeated.
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 37C incubator,
shake the tubes at low speed for 1 hour.
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 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 uL, and load the samples as 8, 4 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.

Latest revision as of 17:16, 26 June 2008

Experiment 1: Cosmic 3760 probe preparation
Step 1: Real time PCR for cosmic probe

Reaction System                                        X8           X12
H2O                                     84.8ul      678.4 ul    1017.6 ul     
10xBuffer                                 10ul         80 ul       120 ul
SYBR  (50x)                              0.5ul          4 ul         6 ul 
*Mix the tube very well using vortex at this point to avoid locally concentrated
SYBR*        
dNTP(10mM)                                 4ul         32 ul        48 ul         
Primer Mix (100uM): Ap1V4IU +Ap2V4       0.4ul        3.2 ul       4.8 ul
template(cosmic3760)                     0.1ul        0.8 ul       1.2 ul 
Jumpstart Taq                            0.2ul        1.6 ul       2.4 ul
 Total                                    100ul        800 ul      1200 ul

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: 8 tubes were prepared and ran first to test if the reaction system is working properly.

 Result: The PCR reaction did not work as the curve never shot up. I am suspecting
 either the Taq enzyme or the 10x buffer is not working, so I will do a comparison to
 test the hypothesis. Since I ran a PCR reaction with Platium Taq and buffer few weeks
 ago, and it worked fine. So it will be used as reference.

Experiment 2: Testing the effectiveness of Jumpstart Taq and buffer

Reaction Systems

 reaction system 1:                                               
 H2O                                     85.1ul           
 10xBuffer                                 10ul               
 dNTP(10mM)                                 4ul                   
 Primer Mix (100uM): Ap1V4IU +Ap2V4       0.4ul           
 SYBR  (50x)                              0.5ul            
 Platium Taq                              0.5ul
 template(cosmic3760)                     0.1ul           
 Total                                    100ul 


 reaction system 2:                                                  
 H2O                                                  78ul      
 10x Platium Taq buffer                               10ul
 Jumpstart Taq polymerase                            0.2ul
 MgCl2(25mM)                                           6ul        
 dNTP(10mM)                                            4ul               
 primer mix (100uM)                                  0.4ul        
 50x SYBG I                                          0.4ul        
 template(cosmic 3760)                               0.1ul       
 Total                                               100ul


Result:

File:6-24-08 cosmic PCR.jpg 


The PCR for both tubes didn't work, thus suggested that the Jumpstart Taq and enzyme were not working properly. However, a control group with platium Taq and buffer was not included, so we couldn't conclude that only the Jumpstart Taq and buffer were causing the problem. Therefore another experiment with control group will be repeated.