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

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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                                       
Reaction Systems                                             
H2O                                    84.8ul       
 
10x Platium Buffer                        10ul      
reaction system 1:                                             
SYBR  (50x)                              0.5ul       
H2O                                    85.6ul         
*Mix the tube very well*
10xBuffer                                10ul              
dNTP(10mM)                                4ul            
dNTP(10mM)                                4ul                  
Primer Mix (100uM): Ap1V4IU +Ap2V4      0.4ul    
Primer Mix (100uM): Ap1V4IU +Ap2V4      0.4ul          
template(cosmic3760)                    0.1ul       
SYBR  (50x)                              0.5ul           
Jumpstart Taq                            0.2ul    
Platium Taq                                1ul
Total                                   100ul
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)                                1ul     
Total                                               100ul

Revision as of 23:37, 24 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.

Reaction Systems

reaction system 1: H2O 85.6ul 10xBuffer 10ul dNTP(10mM) 4ul Primer Mix (100uM): Ap1V4IU +Ap2V4 0.4ul SYBR (50x) 0.5ul Platium Taq 1ul 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) 1ul Total 100ul