Sam:LabNotes/Microbione/2009-3-4/exp2

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Multiplex PCR test - Qiagen kit vs Taq Pol. 2X[edit]

Objective[edit]

  • Test of Qiagen multiplex PCR kit using two group of primers mix.
  • Using Taq Pol.2X as comparison.

Samples & Materials[edit]

  • Qiagen multiplex PCR kit (cat# 206143)
  • Taq 2X master mix (NEB)
  • Templates: 1 ng/uL human gDNA (NEB)


Procedures[edit]

  • Thaw the Qiagen, Taq2X enzyme and primers.
  • Prepare primer mix gouup
    • Primer mix blue: Y-1(122), 21-1(200), 1-1(270), 5-1(413), 22-1(757)
    • Primer mix Yellow: 11-1(131), 10-1(214), 15-1(297), 17-1(419), 16-1(806)
    • Transfer 10 uL from each primer mix stock solution (100 uM for each primer) and mix together -> 20 uM for each primer (total volume: 50 uL)
  • Master Mix - x 2 (Primers mix groups are various)
    • Total reactions: 2 rxns
                             1 rxn x 2       
     -----------------------------
     RNAse-free H2O           15.0 
     Primer mix group (20 uM)  5.0 ---- Primer mix group 1 or 2
     Template (1 ng/uL)        5.0   
     Qiagen kit enzyme (2X)   25.0 
     -----------------------------
                              50.0 (uL) 


                             1 rxn x 2       
     -----------------------------
     RNAse-free H2O           15.0 
     Primer mix group (20 uM)  5.0 ---- Primer mix group 1 or 2
     Template (1 ng/uL)        5.0   
     NEB Taq pol.(2X)         25.0 
     -----------------------------
                              50.0 (uL) 
  • Perform PCR reaction in thermocycler
 Qiagen kit reaction -> QM-PCR52
      Step1   95C, 15m
      Step2   95C, 30s
      Step3   52C, 30s
      Step4   72C, 1m30s
      Step5   Goto Step2 X 35 
      Step6   72C, 10m
      Step7   4C, forever
 Qiagen kit reaction -> QM-PCR52 (The same program used to test individual primer)
      Step1   94C, 2m
      Step2   94C, 40s
      Step3   52C, 40s
      Step4   72C, 1ms
      Step5   Goto Step2 X 35 
      Step6   72C, 10m
      Step7   4C, forever
  • Gel Electrophoresis
    • 1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> 4 small tray with 8-well comb
    • Run at 135 V for 30 min.
    • Loading: Sample 8 uL + 2 uL loading buffer -> 8 uL. Diluted ladder 8 uL
    • Loading order:


Results[edit]

  File:ZhangLab 2 2009-03-04 20hr 52min-run30min.bmp  File:Low mass ladder.bmp
  Left to right: TB -> TY -> QB -> QY -> Low mass ladder
  TB(Taq Pol-Blue primer mix); TY(Taq Pol-Yellow primer mix); QB(Qiagen kit-Blue primer mix); QY(Qiagen kit-Yellow primer mix)
  Primer mix blue: Y-1(122), 21-1(200), 1-1(270), 5-1(413), 22-1(757) 
  Primer mix Yellow: 11-1(131), 10-1(214), 15-1(297), 17-1(419), 16-1(806)
  • Using Taq2X also can performed a good multiplex PCR amplification.
  • Using Qiagen kit got much stronger band which may due to the higher number of amplifying cycles used in the program. The over-amplication in QB and QY lead to saturation problem and one extra band (between 400 and 800 bp).

Suggestion[edit]

  • Using 2% agarose gel to run gel electrophoresis should get better resolution
  • Taq Pol test:
  1. Increase more amplifying cycles.
  2. Use longer extension time to streghten weak bands.
  3. Increase the amount of primers for the weak band genes
  • Qiagen kit test:
  1. Decrease the amplifing cycles.
  2. Try Q-solution provided with the kit.
  • Use higher amount of ladder and load ladder next to the sample well.