MAD confirmation for test 3: Difference between revisions

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>Sam Chiang
>Sam Chiang
 
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*Gel Electrophoresis  
*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
**1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> 2 midium tray with 26-well comb
**Run at 135 V for 20 min.
**Run at 135 V for 20 min.
**Loading: Sample 7 uL + 2 uL Blue juice -> 7 uL. Diluted ladder 7 uL
**Loading: Sample 7 uL + 2 uL Blue juice -> 7 uL. Diluted ladder 7 uL
**Loading order (Left to right): A2->P-Neg (original dilution) -> laddr -> A2->P-Neg (1/10 dilution)
**Loading order (Left to right): A2->P-Neg (original dilution) -> laddr -> A2->P-Neg (1/10 dilution)
 
 


==Results==
==Results==
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*Lower: Test by primer 2-1 (540 bp)
*Lower: Test by primer 2-1 (540 bp)


==Result & Discussion==
 
 
==Discussion==
*Even 1/10 dilutio didn't help improve the regular PCR performance a lot. The p-Pos results still looks very weak in prier 2-1 test
 
*Pick up amplicons from B1 and B4 for 2nd MDA amplification

Latest revision as of 19:59, 2 March 2009

Human cell MDA confirmation for test3[edit]

Objective[edit]

  • PCR test on the 10 candidates MDA amplicons picked up from Test 3.


Samples & Materials[edit]

  • Enzyme - Taq 2X master mix (NEB)
  • Templates (1 uL): A2, A4, A5, A7, B1, B2, B4, B6, B7, C7, D6(p-Pos), D7(p-Neg)-> diluted in 1/10 dilution
  • Primers: 18S-306 (cho. 21, 306 bp, Tm 59C; 2-1 (cho.2, 540 bp, Tm 52C)


Exp. Design[edit]

                                            Templates (A->H)
         ----------------------------------------------------------- 
                A2  A4  A5  A7  B1  B2  B4  B6  B7  C7  p-Pos  p-Neg
         -----------------------------------------------------------
  Primer P1 18S
         P2 2-1 
  Templates were tested in original and 1/10 dilution
        

Procedures[edit]

  • 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 2 (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  


  • Transfer the 1 uL template to PCR tubes (8-well strip)
  • Transfer 9 uL of master mix into each reaction
  • Perform PCR reaction in thermocycler
    • Strip P1 goes to program GENE59 (Tm59, 30 cycles)
    • Strip P2 goes to program GENE52 (Tm52, 30 cycles)
  • Gel Electrophoresis
    • 1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> 2 midium tray with 26-well comb
    • Run at 135 V for 20 min.
    • Loading: Sample 7 uL + 2 uL Blue juice -> 7 uL. Diluted ladder 7 uL
    • Loading order (Left to right): A2->P-Neg (original dilution) -> laddr -> A2->P-Neg (1/10 dilution)


Results[edit]

  File:ZhangLab 2 2009-02-28 17hr 24min-Test3-Primer18S-annotate-new.bmp 
  File:ZhangLab 2 2009-02-28 18hr 12min-Test3-Primer2-1 annotate.bmp
  
  • Upper: Test by primer 18S (306 bp)
  • Lower: Test by primer 2-1 (540 bp)


Discussion[edit]

  • Even 1/10 dilutio didn't help improve the regular PCR performance a lot. The p-Pos results still looks very weak in prier 2-1 test
  • Pick up amplicons from B1 and B4 for 2nd MDA amplification