MAD confirmation for test 3: Difference between revisions

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>Sam Chiang
 
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==Samples & Materials==
==Samples & Materials==


*Enzyme - Taq 2X master mix (New England Biolabs)
*Enzyme - Taq 2X master mix (NEB)  
*Positive control template - 1/100 diluted Jurkat cell genomic DNA (NEB) -> 1 ng/uL
*Templates (1 uL): A2, A4, A5, A7, B1, B2, B4, B6, B7, C7, D6(p-Pos), D7(p-Neg)-> diluted in 1/10 dilution
*Human genome primers
*Primers: 18S-306 (cho. 21, 306 bp, Tm 59C; 2-1 (cho.2, 540 bp, Tm 52C)
**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==
==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)
                                             Templates (A->H)
            ---------------------------------------------------------------------------  
          -----------------------------------------------------------  
                                A3 A6 A7  B4  B7  C3  C4  C8 p-Pos  p-Neg Pos  H2O
                A2 A4  A5 A7 B1  B2 B4 B6 B7  C7 p-Pos  p-Neg
            ---------------------------------------------------------------------------
          -----------------------------------------------------------
   Primer P1 18S-306 (cho. 21, 306 pb, Tm 59C)
   Primer P1 18S
           P2 2-1 (cho.2, 540 bp, Tm 52C)
           P2 2-1  
          P3 3-1 (cho.3, 235 bp, Tm 52C)


  Templates were tested in original and 1/10 dilution
       


==Procedures==
==Procedures==
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*Set up PCR program (GENE59, GENE52).
*Set up PCR program (GENE59, GENE52).


*Master Mix - x 3 (Primers are various)
*Master Mix - x 2 (Primers are various)
**Total reactions: 12 rxns
**Total reactions: 12 rxns


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*Prepare 3 trips of 8-well PCR tubes and transfer the 1 uL template.
*Transfer the 1 uL template to PCR tubes (8-well strip)


*Transfer 9 uL of master mix into each reaction
*Transfer 9 uL of master mix into each reaction


*Perform PCR reaction in thermocycler
*Perform PCR reaction in thermocycler
**Strip P1 goes to block A (program GENE59: Tm59, 30 cycles)
**Strip P1 goes to program GENE59 (Tm59, 30 cycles)
**Strip P2 and P3 go to block B (program GENE52: Tm52, 30 cycles)
**Strip P2 goes to program GENE52 (Tm52, 30 cycles)


*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)




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   [[Image:ZhangLab_2 2009-02-28 test2 primer_18S.bmp|400px]] [[Image:ZhangLab_2 2009-02-28 test2 primer_2-1.bmp|400px]]
   [[Image:ZhangLab_2 2009-02-28 17hr 24min-Test3-Primer18S-annotate-new.bmp|800px]]  
 
  [[Image:ZhangLab_2 2009-02-28 18hr 12min-Test3-Primer2-1 annotate.bmp|800px]]
    
    
*Left: Test by primer 18S (306 bp)   
*Upper: Test by primer 18S (306 bp)   
*Right: 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


*1/100 dilution didn't give us better PCR amplification. Even the p-Pos didn't show up.
*Pick up amplicons from B1 and B4 for 2nd MDA amplification
*The PCR template using MDA amplicon might be used in a range between 1/1 ~ 1/10 dilution.

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