Sam:LabNotes/Microbione/2009-2-25

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Revision as of 20:00, 25 February 2009 by >Sam Chiang (New page: ='''Testing of putative amplification from human cell lysate MDA on 02-24-09'''= ==Objective== *Using regular PCR (Taq enzyme 2X) to confirm four putative amplicons. ==Materials== *...)
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Testing of putative amplification from human cell lysate MDA on 02-24-09

Objective

  • Using regular PCR (Taq enzyme 2X) to confirm four putative amplicons.


Materials

  • Enzyme - Taq 2X master mix (New England Biolabs)
  • Positive control template - 1/100 diluted Jurkat cell genomic DNA (NEB) -> 1 ng/uL
  • Human genome primers
    • 1-1 (cho.1, 270 bp, Tm 52C)
    • 2-1 (cho.2, 540 bp, Tm 52C)
    • 3-1 (cho.3, 235 bp, Tm 52C)
    • 4-1 (cho.4, 383 bp, Tm 52C)
    • 18S-306 (cho. 21) - previously tested
  • Templates (1 uL): B2, B4, B5, C3, E3(p-Neg), Pos(1/100 gDNA), H2O


Exp. Design

                                            Templates (A->H)
           ------------------------------------------------------- 
                                   B2   B4   B5  C4  p-Neg Pos H2O
           -------------------------------------------------------
  Primer   Strip1 P1 (1-1)     
           stirp2 P2 (2-1)    
           strip3 P3 (3-1)
           strip4 P4 (4-1)
           strip5 P5 (18S-306)


Procedures

  • Thaw the Taq2X enzyme and primers.
  • Set up 52C program.
  • Preppare 5 trips of 8-well PCR tubes and transfer the 1 uL template.
  • Master Mix - x 5 tubes:
           1 rxn      8 rxn
     ----------------------
     H2O     3.5       28.0 
     Primer  0.5        4.0  - primer is differnt in each one of five master mix
     Taq 2X  5.0       40.0
     ----------------------
             9.0       72.0 (uL)  72/8=9  
  • Transfer 9 uL of master mix into each reaction
  • Perform PCR reaction
    • Strip 1-4 go to block A (program GENE52: Tm52, 30 cycles)
    • Strip 5 goes to block B (program GENE59: Tm59, 30 cycles)