MAD confirmation for test 3

From ZhangLabWiki
Revision as of 18:01, 2 March 2009 by >Sam Chiang (New page: ='''Testing 1/100 diluted tamplate from human cell MDA amplicon'''= ==Objective== *Test if diluted template of MDA amplcon can perform a better PCR amplification (Better ratio template:p...)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

Testing 1/100 diluted tamplate from human cell MDA amplicon

Objective

  • Test if diluted template of MDA amplcon can perform a better PCR amplification (Better ratio template:primer set or less interference from salts).


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
    • 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

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


                                            Templates (A->H)
           --------------------------------------------------------------------------- 
                                A3  A6  A7  B4  B7  C3  C4  C8  p-Pos  p-Neg  Pos  H2O
           ---------------------------------------------------------------------------
  Primer P1 18S-306 (cho. 21, 306 pb, Tm 59C) 
         P2 2-1 (cho.2, 540 bp, Tm 52C)
         P3 3-1 (cho.3, 235 bp, Tm 52C)


Procedures

  • Thaw the Taq2X enzyme and primers.
  • Dilute all MDA template. Prepare diluted Pos template (1 ng/uL)
  • Set up PCR program (GENE59, GENE52).
  • Master Mix - x 3 (Primers are various)
    • Total reactions: 12 rxns
           1 rxn        12+2 rxn
     ---------------------------
     H2O     3.5       49.0 
     Primer  0.5        7.0  
     Taq 2X  5.0       70.0
     ---------------------------
             9.0      126.0 (uL)  126/14=9  


  • Prepare 3 trips of 8-well PCR tubes and transfer the 1 uL template.
  • Transfer 9 uL of master mix into each reaction
  • Perform PCR reaction in thermocycler
    • Strip P1 goes to block A (program GENE59: Tm59, 30 cycles)
    • Strip P2 and P3 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.
    • Loading: Sample 7 uL + 2 uL Blue juice -> 7 uL. Diluted ladder 7 uL


Results

  File:ZhangLab 2 2009-02-28 test2 primer 18S.bmp File:ZhangLab 2 2009-02-28 test2 primer 2-1.bmp
  
  • Left: Test by primer 18S (306 bp)
  • Right: Test by primer 2-1 (540 bp)


Result & Discussion

  • 1/100 dilution didn't give us better PCR amplification. Even the p-Pos didn't show up.
  • The PCR template using MDA amplicon might be used in a range between 1/1 ~ 1/10 dilution.