Griffitts:5' RACE

Purify 1^st 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 1^st 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.