Alice:LabNotes/2008-6-24: Difference between revisions

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  Reaction System                                        X8          X12
  Reaction System                                        X8          X12
  H2O                                    84.8ul      678.4 ul    1017.6 ul     
  H2O                                    84.8ul      678.4 ul    1017.6 ul     
  10xBuffer                                10ul        80 ul      120 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         
  dNTP(10mM)                                4ul        32 ul        48 ul         
  Primer Mix (100uM): Ap1V4IU +Ap2V4      0.4ul        3.2 ul      4.8 ul
  Primer Mix (100uM): Ap1V4IU +Ap2V4      0.4ul        3.2 ul      4.8 ul
  SYBR  (50x)                             0.5ul          4 ul         6 ul  
  template(cosmic3760)                     0.1ul        0.8 ul       1.2 ul  
  Jumpstart Taq                            0.2ul        1.6 ul      2.4 ul
  Jumpstart Taq                            0.2ul        1.6 ul      2.4 ul
template(cosmic3760)                    0.1ul        0.8 ul      1.2 ul
  Total                                    100ul        800 ul      1200 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 18: Line 20:
94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 22 cycles -> 72C 5min -> 15C hold.<br>
94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 22 cycles -> 72C 5min -> 15C hold.<br>


<b> Note: 8 tubes were prepared and ran first to test if the reaction system is working properly. </b>
Note: 8 tubes were prepared and ran first to test if the reaction system is working properly.  
 
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 Qiaquick columns
 
1. for each 150ul tube, add 750 uL of PBI buffer, mix well, load 450 ul each time to column (twice with total of 900 uL)
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 80 uL ddH2O to each column, wait for 1 min, spin at top speed for 1 min.
9. Pool DNAs from the two tubes into one, measure the concentration with Nanodrop.
 
    * I dissolve the DNA with ddH2O instead of TE to get the system for DnPII incubation.
 
Step 5: Incubation with USER enzyme:
 
Divide the 150ul probe sol into two tubes. In each tube, add 10uL of 10X DpnII reaction buffer and 5 uL of USER (1K/ml)enzyme and last 4 hour on 37C.
Take 4ul out to run gel later.
 
Step 6: Annealing of DpnII oligo and incubation with DpnII (each tube).
 
1. Add 5 uL of 100 uM DpnII guide oligo (SNP and CpG probe need different 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 3 hours at 37C, take 4ul out to run the gel.
5. add 5 uL of USER (1K/ml)enzyme and last another 12 hour. take 4ul out to run the gel.
 
Step 7: Page Denaturing Gel
 
1. Add Novex 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.
!!! Note: starting from this step, everything need to be done in the hood to avoid contamination.!!!
 
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 (100 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.
  Result: The PCR reaction did not work as the curve never shot up. I am suspecting
2. Add 1uL Glycoblue so that one can precipitate better and see the DNA.
  either the Taq enzyme or the 10x buffer is not working, so I will do a comparison to
3. Freeze in -70C for 20 min, spin at 14,000 rpm for 15 min at 4C, discard supernatant with pipette.
  test the hypothesis. Since I ran a PCR reaction with Platium Taq and buffer few weeks
4. Add 500 uL of 75% ethanol to wash DNA, spin at 8000 rpm for 5 min at 4C, discard supernatant with
  ago, and it worked fine. So it will be used as reference.
    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.
<b>Experiment 2: Testing the effectiveness of Jumpstart Taq and buffer</b>


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


1. Add 8uL of 2ng/uL 90-mer and 8 uL of 2x TBE-Urea buffer
  reaction system 1:                                             
  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.
  H2O                                    85.1ul         
3. Denature the DNA with loading dye on thermalcycler 75C for 15 min, then quickly transfer to ice, wait for 1 min.
  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


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.
  reaction system 2:                                                 
  5. Pre-run the gel at 200V for 10 min, then load the samples into the wells, run at 200V for 30 min.  
  H2O                                                  78ul     
    Load the ladder as 8, 4, 2, 1 uL, and load the samples as 10, 5, 2.5 uL.
  10x Platium Taq buffer                              10ul
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.
  Jumpstart Taq polymerase                            0.2ul
    Then quantify the gel to calculate the % yield.
  MgCl2(25mM)                                          6ul       
  dNTP(10mM)                                            4ul             
  primer mix (100uM)                                  0.4ul       
  50x SYBG I                                          0.4ul       
  template(cosmic 3760)                              0.1ul     
  Total                                              100ul
<br>
<b>Result:</b>
 
  [[Image:6-24-08 cosmic PCR.jpg]]
<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.

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.