Sam:LabNotes/Microbione/2009-3-2: Difference between revisions

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='''Human genome screening primer set - quality validation'''=


=Objective=
*Human whole genome primer sets were obtained from Professor (2 sets).
*The primer set were used to confirm the existence of chromosomes in MDA amplicon using multiplex PCR.
*Individual validation of each primer is required before performing multiplex PCR.
==Samples & Materials==
*Enzyme - Taq 2X master mix (NEB)
*Templates (1 uL): 1 ng/uL human gDNA (NEB)
*Primers: All primers are diluted to 100 uM, and mixed basd on the gene (Forward 20 uL + Reverse 20 uL + H2O 160 uL)
**Set1: 1-1 ~ 22-1 and X-1, Y-1 (24 rxns)
**Set2: 1-2 ~ 22-2 and X-2 (23 rxns)
                 
==Procedures==
*Thaw the Taq2X enzyme and primers.
*Dilute all MDA template. Prepare diluted Pos template (1 ng/uL)
*Set up PCR program (GENE59, GENE52).
*Master Mix - x 2 (Primers are various)
**Total reactions: 12 rxns
            1 rxn        24+3 rxn
      ---------------------------
      H2O      3.5      94.5
      Template 1.0      27.0 
      Taq 2X  5.0      135.0
      ---------------------------
              9.5      256.5 (uL)  256.5/27=9.5
*Transfer 9.5 uL of master mix into each of 24 PCR tubes (8-well strip)
*Transfer the 0.5 uL primer into each reaction
*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 -> 2 medium tray with 26-well comb
**Run at 135 V for 18 min.
**Loading: Sample 7 uL + 2 uL loading buffer -> 7 uL. Diluted ladder 7 uL
*Repeat the same design to perform PCR on primer set2 (23 reactions)
==Results==
  [[Image:ZhangLab_2 2009-03-03 15hr 01min-set1-new.jpg|600px]]
  [[Image:ZhangLab_2 2009-03-03 15hr 01min-set2.jpg|600px]]
 
*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==
*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

Latest revision as of 20:43, 8 March 2009