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

From ZhangLabWiki
Jump to navigation Jump to search

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