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:
- Increase more amplifying cycles.
- Use longer extension time to streghten weak bands.
- Increase the amount of primers for the weak band genes
- Qiagen kit test:
- Decrease the amplifing cycles.
- Try Q-solution provided with the kit.
- Use higher amount of ladder and load ladder next to the sample well.