Griffitts:5' RACE

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

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

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
Note: A third round with yet another nested primer may be necessarybr>
 * Use 54°C annealing temperature
 * Use nested primer and poly-T primer (oJG636)
 * Use template from 1st round of PCR

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.