Haynes Lab:Notebook/Jan/2015/12/16

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RT-PCR for HOXD11/HOXD12/GAPDH on U2OS and iPS cells

REACTION LIST
  Template cDNA Gene Target
Rxn 1: U2OS cells HOXD11 (1)
Rxn 2: U2OS cells HOXD11 (2)
Rxn 3: U2OS cells HOXD11 (3)
Rxn 4: U2OS cells HOXD12 (1)
Rxn 5: U2OS cells HOXD12 (2)
Rxn 6: U2OS cells GAPD
Rxn 7: iPS cells HOXD11 (1)
Rxn 8: iPS cells HOXD11 (2)
Rxn 9: iPS cells HOXD11 (3)
Rxn 10: iPS cells HOXD12 (1)
Rxn 11: iPS cells HOXD12 (2)
Rxn 12: iPS cells GAPD
Rxn 13: no template HOXD11 (1)
Rxn 14: no template HOXD11 (2)
Rxn 15: no template HOXD11 (3)
Rxn 16: no template HOXD12 (1)
Rxn 17: no template HOXD12 (2)
Rxn 18: no template GAPD

Plate Layout

Image:Jan_Simper_12_16_15_Plate_Layout.png

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

GAPD: GAPD primers, GAPD probe

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
  • GAPD - 85.0 μL

Master mix for each template

First, make a 1:10 dilution of the cDNA.

One cell type is in 6 reactions, 3 replicates + 1 pipetting error.

6 * 3 + 1 = 19

Reagent (Single well) cDNA Template (x19)
1:10 cDNA dilution (2.0 μL) 38.0*
PCR H2O (4.5 μL) 85.5
Total vol. (6.5 μL) 123.5

*For the no template control, use PCR H2O instead of cDNA.

Resulting 1.5 mL tubes:

  • U2OS - U2OS cells cDNA, 123.5 μL
  • iPS - iPS cells cDNA, 123.5 μL
  • Con - no template control, 123.5 μL


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