Sam:LabNotes/Human/2009-2-25 exp1

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Testing of putative amplification from human cell lysate MDA on 02-24-09[edit]

Objective[edit]

  • Using regular PCR (Taq enzyme 2X) to confirm four 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
    • 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[edit]

                                            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[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      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)
  • Gel Electrophoresis
    • 1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe)
    • Run at 135 V for 30 min.


Results[edit]

FIG. Gel electrophoresis picture

  File:ZhangLab 2 2009-02-26 09hr 46min copy.bmp File:Low mass ladder.bmp
  Left gel: test by primer 2-1 (540 bp); Right gel: test by primer 3-1 (235 bp)
  File:ZhangLab 2 2009-02-26 12hr 04min.bmp
  Left gel: test by primer 4-1 (383 bp); Right gel: test by primer 18S (306 bp)

Discussion[edit]

  • None of four putative polonies has contained the human genomic DNA
  • The n-Neg (Negitive control in MDA) has no band, which means the contamination is not human genome either.
  • The 10 uL reaction volume works fine for this applification (PCR testing) and is able to save more enzyme for multiple reaction.
  • The ladder is not shown well on 100, 200, 300 bp band even 6 uL of ladder was used.


Suggestion[edit]

  • For gel electrophoresis, running at 135V, 20 min is sufficient.
  • Need to repeat the MDA using different scale of dilution method and try to decrease the background in MDA reaction.
  • It is also possible the salts left in MDA reaction inhibited the following ragular PCR reaction (PCR products need to be purified before they can serves as new templates).