Noi:LabNotes/2010-10-21

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Revision as of 16:24, 23 October 2010 by >Noi
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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 15 cycles
   60C 2min  
   15C hold.
   
   - Ethanol precipitation
   - Re-purified with twelve Qiaquick columns
   - Each eluted with 41ul EB (total vol: 12 x 41 = 492ul).
                                            DMR220k_S4                             DMR220k_S5
  Amplified amplicon conc. (ng/ul)            194.2                                    192.7                                                
  O.D.260/280                                 1.93                                     1.92                             
  Yield: conc. x total vol. 194.2ng/ulx492ul= 95.5ug                192.7ng/ulx492ul = 94.8ug

Lambda exo digestion

                                            DMR220k_S4                             DMR220k_S5
    Qiaquick (QQ)amplified amplicon           108ul (21.0ug)                            108ul (20.8ug)
    10x Lambda Exo buffer                     15ul                                      15ul
    Lambda exo                                10ul                                      10ul
    H20                                       17ul                                      17ul
                                              =====================================================
    Total                                     100ul x 4.5                               100ul x 4.5
    Total amount of QQ amplified amplicon     94.4ug                                    93.6ug
  
   - 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)    81.3                                      75.9                                                    
   O.D.260/280                                 1.96                                      1.96                                
   Yield: conc. x total vol. 81.3ng/ulx240ul = 19.5ug                  75.9ng/ulx240ul = 18.2ug

Removal of amplification adaptors

                                            DMR220k_S4                             DMR220k_S5    
  ss-amplicon                                  75ul (6.1ug)                               75ul (5.7ug)
  USER                                         5ul                                        5ul
                                               =======================================================
  Total                                        80ul x 3                                   80ul x 3
  Total amount of ss amplicon                  18.3ug                                     17.1ug
  - 37C 1hr
  - Add 
        10x DpnII buffer                       10ul                                       10ul
        100uM RE-DpnII-V66 guide oligo         5ul                                        5ul
  - 94C 2min, 37C 3min
  - Add DpnII                                  5ul                                        5 ul
  - 37C 3hr
  - Add 
        USER                                   5ul                                        5ul
        10x DpnII buffer                       10ul                                       10ul
        H2O                                    85ul                                       85ul
  - 37C O/N