Kun:LabNotes/HiResChrPaint/2012-4-4: Difference between revisions

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m (Created page with '==Processing of oligo pools (STEP 1)== *Received the oligo pools from Mycorarray in four tubes. These are "ssDNA from emulsion PCR pool". Resuspend each pool in 500ul nuclease fr…')
 
Line 7: Line 7:
     5435_ssDNA  80k_set_3  ~2.8ug  85pmole    170nM      50ul+374ul H2O => 20nM
     5435_ssDNA  80k_set_3  ~2.8ug  85pmole    170nM      50ul+374ul H2O => 20nM
*There is probably no need to do expansion PCR since the oligo pools were already amplified by emulsion PCR.
*There is probably no need to do expansion PCR since the oligo pools were already amplified by emulsion PCR.
===Confirmation PCR===
    V6 pool
                                Each   
    20nM oligo pool              0.2ul   
    2x Kapa SYBG qPCR Master Mix  50ul   
    100uM pAP1V61U              0.4ul   
    100uM AP2V6                  0.4ul   
    2mM aa-dUTP                    3ul     
    H2O                          41ul   
    ===================================
                                100ul
    95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold.
    V4 pool
                                Each   
    20nM oligo pool              0.2ul 
    2x Kapa SYBG qPCR Master Mix  50ul
    100uM pAP1V41U              0.4ul 
    100uM AP2V4                  0.4ul 
    2mM aa-dUTP                    3ul   
    H2O                          41ul 
    ===================================
                                100ul
    95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.
    Check the size distribution of the amplicons with 6% TBE gels.
===Production PCR===
===Production PCR===
     V6 pool for all four sets
     V6 pool for all four sets
Line 14: Line 39:
     100uM pAP1V61U              0.4ul    6.4
     100uM pAP1V61U              0.4ul    6.4
     100uM AP2V6                  0.4ul    6.4
     100uM AP2V6                  0.4ul    6.4
     H2O                          44ul   704
    2mM aa-dUTP                    3ul    48 
     H2O                          41ul   656
     ===================================
     ===================================
                                 100ul  
                                 100ul  
Line 25: Line 51:
     100uM pAP1V41U              0.4ul    6.4
     100uM pAP1V41U              0.4ul    6.4
     100uM AP2V4                  0.4ul    6.4
     100uM AP2V4                  0.4ul    6.4
     H2O                          44ul   704
    2mM aa-dUTP                    3ul    48 
     H2O                          41ul   656
     ===================================
     ===================================
                                 100ul x 16
                                 100ul x 16
     95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.
     95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.
    Check the size distribution of the amplicons with 6% TBE gels.
    Ethanol precipitation.
    Re-purified each amplicon with two Qiaquick columns, eluted with 40ul EB.
    Measure concentration.
===Adaptor removal===
*Dpn II digestion.
    ds-oligo pools    75ul
    10X Dpn II buffer 10ul
    Dpn II (10U/ul)    8ul  (might need to adjust the amount of enzyme based on the yield of PCR)
    H2O                7ul
    37C 30min -> 65C 20min -> Qiaquick column purification
*Lambda exo digestion.
    Dpn II digested oligo pool    40ul
    10X Lambda Exo buffer        10ul
    Lambda Exo (10U/ul)          10ul
    H2O                          40ul
    37C 1h -> 75C 10min -> Qiaquick column purification
*Proceed with AlexaFluor coupling.

Revision as of 23:29, 4 April 2012

Processing of oligo pools (STEP 1)

  • Received the oligo pools from Mycorarray in four tubes. These are "ssDNA from emulsion PCR pool". Resuspend each pool in 500ul nuclease free H2O.
  • Take 50ul of each oligo pool, dilute to 20nM. These will be the working tubes.
   5431_ssDNA  80k_set_4   ~2.8ug   85pmole     170nM      50ul+374ul H2O => 20nM
   5432_ssDNA  80k_set_1   ~1.8ug   55pmole     109nM      50ul+223ul H2O => 20nM
   5433_ssDNA  80k_set_2   ~1.7ug   52pmole     103nM      50ul+208ul H2O => 20nM
   5435_ssDNA  80k_set_3   ~2.8ug   85pmole     170nM      50ul+374ul H2O => 20nM
  • There is probably no need to do expansion PCR since the oligo pools were already amplified by emulsion PCR.

Confirmation PCR

   V6 pool
                                Each     
   20nM oligo pool              0.2ul    
   2x Kapa SYBG qPCR Master Mix  50ul    
   100uM pAP1V61U               0.4ul    
   100uM AP2V6                  0.4ul    
   2mM aa-dUTP                    3ul       
   H2O                           41ul    
   ===================================
                                100ul 
   95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold.
   V4 pool
                                Each    
   20nM oligo pool              0.2ul  
   2x Kapa SYBG qPCR Master Mix  50ul
   100uM pAP1V41U               0.4ul  
   100uM AP2V4                  0.4ul   
   2mM aa-dUTP                    3ul     
   H2O                           41ul  
   ===================================
                                100ul 
   95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.
   Check the size distribution of the amplicons with 6% TBE gels.

Production PCR

   V6 pool for all four sets
                                Each     x16
   20nM oligo pool              0.2ul    3.2
   2x Kapa SYBG qPCR Master Mix  50ul    800
   100uM pAP1V61U               0.4ul    6.4
   100uM AP2V6                  0.4ul    6.4
   2mM aa-dUTP                    3ul     48  
   H2O                           41ul    656
   ===================================
                                100ul 
   95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold.
   V4 pool for sets #1, #3, #4
                                Each     x16
   20nM oligo pool              0.2ul    3.2
   2x Kapa SYBG qPCR Master Mix  50ul    800
   100uM pAP1V41U               0.4ul    6.4
   100uM AP2V4                  0.4ul    6.4
   2mM aa-dUTP                    3ul     48  
   H2O                           41ul    656
   ===================================
                                100ul x 16
   95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold.
   Check the size distribution of the amplicons with 6% TBE gels.
   Ethanol precipitation.
   Re-purified each amplicon with two Qiaquick columns, eluted with 40ul EB.
   Measure concentration.

Adaptor removal

  • Dpn II digestion.
   ds-oligo pools    75ul 
   10X Dpn II buffer 10ul
   Dpn II (10U/ul)    8ul  (might need to adjust the amount of enzyme based on the yield of PCR)
   H2O                7ul
   37C 30min -> 65C 20min -> Qiaquick column purification
  • Lambda exo digestion.
   Dpn II digested oligo pool    40ul
   10X Lambda Exo buffer         10ul
   Lambda Exo (10U/ul)           10ul
   H2O                           40ul
   37C 1h -> 75C 10min -> Qiaquick column purification
  • Proceed with AlexaFluor coupling.