Haynes Lab:Notebook/Jan/2016/04/13

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RT-PCR Check of HoxD11/12 Primers

REACTION LIST
  Template cDNA Gene Target
Rxn 1: HOXD Amplicons 1 μL HOXD11 (1)
Rxn 2: HOXD Amplicons 1 μL HOXD11 (2)
Rxn 3: HOXD Amplicons 1 μL HOXD11 (3)
Rxn 4: HOXD Amplicons 1 μL HOXD12 (1)
Rxn 5: HOXD Amplicons 1 μL HOXD12 (2)
Rxn 6: HOXD Amplicons 2 μL HOXD11 (1)
Rxn 7: HOXD Amplicons 2 μL HOXD11 (2)
Rxn 8: HOXD Amplicons 2 μL HOXD11 (3)
Rxn 9: HOXD Amplicons 2 μL HOXD12 (1)
Rxn 10: HOXD Amplicons 2 μL HOXD12 (2)
Rxn 11: HOXD Amplicons 3 μL HOXD11 (1)
Rxn 12: HOXD Amplicons 3 μL HOXD11 (2)
Rxn 13: HOXD Amplicons 3 μL HOXD11 (3)
Rxn 14: HOXD Amplicons 3 μL HOXD12 (1)
Rxn 15: HOXD Amplicons 3 μL HOXD12 (2)

Primers

HOXD11 (1): HOXD11 L1, HOXD11 R1, UPL Probe 18

HOXD11 (2): HOXD11 L2, HOXD11 R2, UPL Probe 60

HOXD11 (3): HOXD11 L3, HOXD11 R3, UPL Probe 1

HOXD12 (1): HOXD12 L1, HOXD12 R1, UPL Probe 70

HOXD12 (2): HOXD12 L2, HOXD12 R2, UPL Probe 84

Mixes

PCR master mix for each gene target.

One gene target is in 3 reactions, and 3 replicates per reaction, +1 for pipetting error.

3 x 3 + 1 = 10

Reagent (Single well) Gene Target (x10) GAPD (x10)
2x LC480 Probes Master (7.5 μL) 75.0 75.0
20 μM Forward primer (0.3 μL) 3.0 3.0 GAPD primers*
20 μM Reverse primer (0.3 μL) 3.0 ---
10 μM UPL probe (0.3 μL) 3.0 3.0 GAPD UPL probe*
PCR H2O (0.1 μL) 1.0 4.0
Total vol. (8.5 μL) 85.0 85.0

Resulting 1.5 mL tubes:

  • HOXD11 (1) - 85.0 μL
  • HOXD11 (2) - 85.0 μL
  • HOXD11 (3) - 85.0 μL
  • HOXD12 (1) - 85.0 μL
  • HOXD12 (2) - 85.0 μL

Master mix for each template

First, dilute the 2453 fmol of HOXD Amplicons to 20 fmol/μL by adding 122.65 μL of PCR H2O

One cell type is in 5 reactions, 3 replicates + 1 pipetting error.

5 * 3 + 1 = 16

Reagent (Single well) cDNA Template (x16)
HOXD Amplicons (1.0 μL) 16.0*
PCR H2O (5.5 μL) 88
Total vol. (6.5 μL) 104
Reagent (Single well) cDNA Template (x16)
HOXD Amplicons (2.0 μL) 32.0*
PCR H2O (4.5 μL) 72
Total vol. (6.5 μL) 104
Reagent (Single well) cDNA Template (x16)
HOXD Amplicons (3.0 μL) 48.0*
PCR H2O (3.5 μL) 56
Total vol. (6.5 μL) 104

Resulting 1.5 mL tubes:

  • 1 μL - 20 fmol/uL HOXD Amplicon DNA, 104 μL
  • 2 μL - 40 fmol/uL HOXD Amplicon DNA, 104 μL
  • 3 μL - 60 fmol/uL HOXD Amplicon DNA, 104 μL


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