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

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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.  


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.
  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.


Reaction System                                       
<b>Experiment 2: Testing the effectiveness of Jumpstart Taq and buffer</b>
H2O                                    84.8ul       
 
10x Platium Buffer                        10ul      
Reaction Systems                                             
SYBR  (50x)                              0.5ul       
 
*Mix the tube very well*
  reaction system 1:                                             
dNTP(10mM)                                4ul            
  H2O                                    85.1ul         
Primer Mix (100uM): Ap1V4IU +Ap2V4      0.4ul    
  10xBuffer                                10ul              
template(cosmic3760)                    0.1ul       
  dNTP(10mM)                                4ul                  
Jumpstart Taq                            0.2ul    
  Primer Mix (100uM): Ap1V4IU +Ap2V4      0.4ul          
Total                                   100ul
  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
<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.