Alice:LabNotes/2008-6-24

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Revision as of 23:38, 24 June 2008 by >Zsakura2
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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