Sam:LabNotes/Human/2009-2-26

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Testing of putative amplification from human cell lysate MDA test 2 (on 02-25-09)[edit]

Objective[edit]

  • Using regular PCR (Taq enzyme 2X) to confirm 8 putative amplicons.


Materials[edit]

  • 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
    • 18S-306 (cho. 21) - previously tested
    • 2-1 (cho.2, 540 bp, Tm 52C)
    • 3-1 (cho.3, 235 bp, Tm 52C)


  • Templates (1 uL): A3, A6, A7, B4, B7, C3, C4, C8, D6(p-Pos), D7(p-Neg), Pos(1/100 gDNA = 1 ng/uL), H2O


Exp. Design[edit]

                                            Templates (A->H)
           --------------------------------------------------------------------------- 
                                A3  A6  A7  B4  B7  C3  C4  C8  p-Pos  p-Neg  Pos  H2O
           ---------------------------------------------------------------------------
  Primer   Strip1 P1 (18S-306)     
           stirp2 P2 (2-1)    
           strip3 P3 (3-1)

Procedures[edit]

  • 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      14 rxn
     ----------------------
     H2O     3.5       49.0 
     Primer  0.5        7.0  - primer is differnt in each one of three master mix
     Taq 2X  5.0       70.0
     ----------------------
             9.0      126.0 (uL)  126/14=9  
  • Transfer 9 uL of master mix into each reaction
  • Perform PCR reaction
    • Strip 1 goes to block A (program GENE59: Tm59, 30 cycles)
    • Strip 2-3 go to block B (program GENE52: Tm52, 30 cycles)
  • Gel Electrophoresis
    • 1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> four small tray with 8-well comb)
    • Run at 135 V for 20 min.

Results[edit]

FIG. Gel electrophoresis picture

  File:ZhangLab 2 2009-02-26 test2 primer 18S.bmp File:Low mass ladder.bmp
  Test by primer 18S (306 bp)
  File:ZhangLab 2 2009-02-26 test2 primer 2-1.bmp
  Test by primer 2-1 (540 bp)
  File:ZhangLab 2 2009-02-26 test2 primer 3-1.bmp
  Test by primer 3-1 (235 bp)

Result & Discussion[edit]

  • Samle B7 and p-Pos contains positive bands by 18S primer (Cho.21/Un) and primer 3-1 (Cho.3).
  • A very weak band was also found (but not showing clearly in pic) on B7 by primer 2-1 (Cho.2). However the p-Pos failed to show it.
    • Sample p-Pos should contain successful amplification. The faliure of p-Pos may be caused by the interference of junk (salt?) left in MDA PCR product.
    • Clean the junk using PCR product purification kit and test the PCR again on those p-Pos sample.