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='''Multiplex PCR test - Qiagen kit vs Taq Pol. 2X'''= ==Objective== *Test of Qiagen multiplex PCR kit using two group of primers mix. *Using Taq Pol.2X as comparison. ==Samples & Materials== *Qiagen multiplex PCR kit (cat# 206143) *Taq 2X master mix (NEB) *Templates: 1 ng/uL human gDNA (NEB) ==Procedures== *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== [[Image:ZhangLab_2 2009-03-04 20hr 52min-run30min.bmp|350px]] [[Image:low mass ladder.bmp|150px]] '''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== *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.
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