Griffitts:5' RACE

From OpenWetWare

Revision as of 13:30, 11 April 2008 by Matthew B. Crook (Talk | contribs)
(diff) ←Older revision | Current revision (diff) | Newer revision→ (diff)
Jump to: navigation, search

Contents

Purify 1st strand DNA

  • Place completed RT reaction at 100°C for 2 minutes
  • Place on ice
  • Add 1.5 μL RNAse A
  • Place at 37°C for 10 minutes
  • Add 200 μL buffer PB into sample
  • Move to column
  • Spin 30 seconds
  • Remove flow-through
  • Add 500 μL buffer PE to column
  • Spin 30 seconds
  • Remove flow-through
  • Spin an additional 60 seconds to remove residual PE buffer
  • Move column to new 1.5-mL tube
  • Add 35 μL TE Buffer
  • Let stand for 1 minute
  • Spin 1 minute

Adding poly-A tail

Reaction recipe

  • 7 μL ddH2O
  • 7 μL purified 1st strand DNA
  • 2 μL NE Buffer 4
  • 2 μL cobalt chloride (solution provided with TdT enzyme)
  • 1 μL 2 mM dATP (98 μL ddH2O + 2 μL dATP)
  • 1 μL TdT (in freezer; keep on ice)

Procedure

  • Combine
  • Incubate at 37°C for 30 minutes
  • Store in –20°C freezer

PCR

Reaction recipe

  • 35.4 μL ddH2O
  • 5 μL Taq buffer
  • 1.2 μL dNTP
  • 0.4 μL Taq polymerase
  • 2 μL template
  • 3 μL 10 μM primer
  • 3 μL 10 μM primer

Round One

  • Use 48°C annealing temperature
  • Use poly-T primer (oJG636) and outside gene-specific primer

Round Two

  • Use 54°C annealing temperature
  • Use nested primer and poly-T primer (oJG636)
  • Use template from 1st round of PCR

Note: A third round with yet another nested primer may be necessarybr>

When ready, proceed to gel electrophoresis.

oJG636

CGCGGATCCTCTAGATTTTTTTTTTTTTTTTTT
Note: this oligo-dT primer contains additional GC-rich sequence (BamHI/XbaI sites) at its 5’ end.

Personal tools