Sam:LabNotes/Microbione/2009-3-2: Difference between revisions

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>Sam Chiang
(New page: =Human genome screening primer set - quality validation= ==Objective== *Human whole genome primer sets were obtained from Professor (2 sets). *The primer set were used to confirm the exi...)
 
>Sam Chiang
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**Set1: 1-1 ~ 22-1 and X-1, Y-1 (24 rxns)
**Set1: 1-1 ~ 22-1 and X-1, Y-1 (24 rxns)
**Set2: 1-2 ~ 22-2 and X-2 (23 rxns)
**Set2: 1-2 ~ 22-2 and X-2 (23 rxns)
       
                 
 
==Exp. Design==
         
                                            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==
==Procedures==
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**Total reactions: 12 rxns
**Total reactions: 12 rxns


             1 rxn        12+2 rxn
             1 rxn        24+3 rxn
       ---------------------------
       ---------------------------
       H2O    3.5      49.0  
       H2O    3.5      94.5
       Primer  0.5        7.0   
    Template 1.0      27.0   
       Taq 2X  5.0       70.0
       Taq 2X  5.0     135.0
       ---------------------------
       ---------------------------
               9.0     126.0 (uL)  126/14=9
               9.5     256.5 (uL)  256.5/27=9.5




*Transfer the 1 uL template to PCR tubes (8-well strip)
*Transfer 9.5 uL of master mix into each of 24 PCR tubes (8-well strip)
 
*Transfer the 0.5 uL primer into each reaction
*Transfer 9 uL of master mix into each reaction
*Perform PCR reaction in thermocycler: program GENE52 (Tm52, 30 cycles)
 
*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  
*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
**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 18 min.
**Loading: Sample 7 uL + 2 uL Blue juice -> 7 uL. Diluted ladder 7 uL
**Loading: Sample 7 uL + 2 uL loading buffer -> 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): 1-1 -> 22-1 -> X-1 ->Y-1 -> Low mass ladder


*Repeat the same design to perform PCR on primer set2 (23 reactions)




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   [[Image:ZhangLab_2 2009-02-28 17hr 24min-Test3-Primer18S-annotate-new.bmp|800px]]  
   [[Image:ZhangLab_2 2009-03-03 15hr 01min-set1.jpg|800px]]  


   [[Image:ZhangLab_2 2009-02-28 18hr 12min-Test3-Primer2-1 annotate.bmp|800px]]
   [[Image:ZhangLab_2 2009-03-03 15hr 01min-set2.jpg|800px]]
    
    
*Upper: Test by primer 18S (306 bp)   
*Upper: Test by primer 18S (306 bp)   
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==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

Revision as of 07:30, 5 March 2009

Human genome screening primer set - quality validation

Objective

  • Human whole genome primer sets were obtained from Professor (2 sets).
  • The primer set were used to confirm the existance of choromosomes in MDA amplicon using multiplex PCR.
  • Individual validation of each primer is required before performing multiplex PCR.


Samples & Materials

  • Enzyme - Taq 2X master mix (NEB)
  • Templates (1 uL): 1 ng/uL human gDNA (NEB)
  • Primers: All primers are diluted to 100 uM, and mixed basd on the gene (Forward 20 uL + Reverse 20 uL + H2O 160 uL)
    • Set1: 1-1 ~ 22-1 and X-1, Y-1 (24 rxns)
    • Set2: 1-2 ~ 22-2 and X-2 (23 rxns)


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 2 (Primers are various)
    • Total reactions: 12 rxns
           1 rxn        24+3 rxn
     ---------------------------
     H2O     3.5       94.5
    Template 1.0       27.0  
     Taq 2X  5.0      135.0
     ---------------------------
             9.5      256.5 (uL)  256.5/27=9.5 


  • Transfer 9.5 uL of master mix into each of 24 PCR tubes (8-well strip)
  • Transfer the 0.5 uL primer into each reaction
  • Perform PCR reaction in thermocycler: 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 18 min.
    • Loading: Sample 7 uL + 2 uL loading buffer -> 7 uL. Diluted ladder 7 uL
    • Loading order (Left to right): 1-1 -> 22-1 -> X-1 ->Y-1 -> Low mass ladder
  • Repeat the same design to perform PCR on primer set2 (23 reactions)


Results

  File:ZhangLab 2 2009-03-03 15hr 01min-set1.jpg 
  File:ZhangLab 2 2009-03-03 15hr 01min-set2.jpg
  
  • Upper: Test by primer 18S (306 bp)
  • Lower: Test by primer 2-1 (540 bp)


Discussion