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

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===Production PCR===
===Production PCR===
                                                                     
 
     10nM 1st round amplicon      0.2ul                 
     10nM 1st round amplicon      0.2ul                 
     2x Kapa SYBG qPCR Master Mix  50ul         
     2x Kapa SYBG qPCR Master Mix  50ul         
Line 14: Line 14:
     95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 16 -> 60C 2min -> 15C hold.
     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).
     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
  Amplified amplicon conc. (ng/ul)          93.2                                    92.5                                                 
  O.D.260/280                              1.93                                    1.92                           
  Yield: conc. x total vol.                2.35                                    2.35


                                                                                          DMR220k_S4                                            DMR220k_S5
===Lambda exo digestion===


  Qiaquick amplified amplicon (approx. 20ug)
      DMR220k_S4                                            DMR220k_S5
  10x Lambda Exo buffer
    Qiaquick amplified amplicon (approx. 20ug)     16    15
  Lambda exo
    10x Lambda Exo buffer
  H20  
    Lambda exo
    H20  
    
    
-> 37C 1h -> purified with four Qiaquick columns, each eluted with 60ul EB (total vol: 4 x 60 = 240ul).
-> 37C 1h -> purified with four Qiaquick columns, each eluted with 60ul EB (total vol: 4 x 60 = 240ul).

Revision as of 05:39, 23 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)          93.2                                    92.5                                                  
  O.D.260/280                               1.93                                    1.92                             
  Yield: conc. x total vol.                 2.35                                    2.35

Lambda exo digestion

     DMR220k_S4                                             DMR220k_S5
    Qiaquick amplified amplicon (approx. 20ug)      16    15
    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.