Sam:LabNotes/Microbione/2009-3-3/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: program GENE52 (Tm52, 30 cycles)
- 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
- Repeat the same design to perform PCR on primer set2 (23 reactions)
Results[edit]
File:ZhangLab 2 2009-03-03 15hr 01min-set1-new.jpg
File:ZhangLab 2 2009-03-03 15hr 01min-set2.jpg
- Upper: Test by primer set1 (1-1 -> 8-1, ladder, 9-1 -> 16-1, ladder, 17-1 -> 22-1, X-1, Y-1)
- Lower: Test by primer set2 (1-2 -> 8-2, ladder, 9-2 -> 16-2, ladder, 17-2 -> 22-2, X-2)
- Primer name (amplicon size)
1-1 (270) 4-1 (383) 7-1 (242) 10-2 (299) 13-2 (1102) 16-2 (1324) 19-1 (1007) 22-2 (1166) 1-2 (676) 4-2 (896) 7-2 (498) 10-1 (214) 13-1 (679) 16-1 (806) 19-2 (1512) 22-1 (757) 2-1 (540) 5-1 (413) 8-2 (231) 11-1 (131) 14-1 (598) 17-2 (1240) 20-1 (174) x-2 (839) 2-2 (649) 5-2 (530) 8-1 (230) 11-2 (525) 14-2 (756) 17-1 (419) 20-2 (584) x-1 (147) 3-1 (235) 6-2 (1050) 9-1 (373) 12-1 (984) 15-1 (297) 18-2 (288) 21-1 (200) y-1 (122) 3-2 (1323) 6-1 (299) 9-2 (907) 12-2 (1689) 15-2 (1422) 18-1 (171) 21-2 (346)
Discussion & Suggestion[edit]
- Except primer set1 chromosome X and Y which have only weak bands, all primers generate strong band with good amplification at 52C
- The PCR cycle is too many -> saturation of amplicons
- The primer concentration used in PCR is too high -> serious primer dimer seen on the gel