Noi:LabNotes/2010-10-21: Difference between revisions

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                                                                        DMR220k_S4                                            DMR220k_S5
                                                                                        DMR220k_S4                                            DMR220k_S5
     DNA concentration (ng/ul)                                                     
     Amplified amplicon conc. (ng/ul)                                                     
     O.D.260/280                                     
     O.D.260/280                                     
     Yield: concxtotal vol.
     Yield: conc. x total vol.
 
===Lambda exo digestion=== 
 
                                                                                          DMR220k_S4                                            DMR220k_S5
 
  Qiaquick amplified amplicon (approx. 20ug)
  10x Lambda Exo buffer
  Lambda exo
  H20
 
-> 37C 1h -> purified with four Qiaquick columns, each eluted with 60ul EB (total vol: 4 x 60 = 240ul).
 
                                                                                          DMR220k_S4                                            DMR220k_S5
    Single-strand(SS) amplicon conc. (ng/ul)                                                   
    O.D.260/280                                   
    Yield: conc. x total vol.

Revision as of 15:21, 22 October 2010

Probe prep for the DMR220k_S4 and S5

(followed Dr. Zhang's conditions)

Production PCR

   10nM 1st round amplicon      0.2ul                
   2x Kapa SYBG qPCR Master Mix  50ul        
   100uM pAP1V61U               0.4ul                  
   100uM AP2V6                  0.4ul                      
   H2O                           49ul                          
   ===================================
                                100ul x 96
   95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 16 -> 60C 2min -> 15C hold.
   Ethanol precipitation; re-purified with twelve Qiaquick columns, each eluted with 40ul EB (total vol: 12 x 40 = 480ul).
   
                                                                                        DMR220k_S4                                             DMR220k_S5
   Amplified amplicon conc. (ng/ul)                                                     
   O.D.260/280                                     
   Yield: conc. x total vol.

Lambda exo digestion

                                                                                          DMR220k_S4                                             DMR220k_S5
  Qiaquick amplified amplicon (approx. 20ug)
  10x Lambda Exo buffer
  Lambda exo
  H20 
  

-> 37C 1h -> purified with four Qiaquick columns, each eluted with 60ul EB (total vol: 4 x 60 = 240ul).

                                                                                          DMR220k_S4                                             DMR220k_S5
   Single-strand(SS) amplicon conc. (ng/ul)                                                     
   O.D.260/280                                     
   Yield: conc. x total vol.