Griffitts:5' RACE: Difference between revisions
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==Purify 1<sup>st</sup> strand DNA== | ==Purify 1<sup>st</sup> strand DNA== | ||
* Place [[ | * Place [[Griffitts:Reverse transcription|completed RT reaction]] at 100°C for 2 minutes | ||
* Place on ice | * Place on ice | ||
* Add 1.5 μL [[Griffitts:Common buffers#RNAse A|RNAse A]] | * Add 1.5 μL [[Griffitts:Common buffers#RNAse A|RNAse A]] | ||
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===Reaction recipe=== | ===Reaction recipe=== | ||
* 7 μL ddH<sub>2</sub>O | * 7 μL ddH<sub>2</sub>O | ||
* 7 μL [[Griffitts:5'RACE#Purify 1st strand DNA|purified 1<sup>st</sup> strand DNA]] | * 7 μL [[Griffitts:5' RACE#Purify 1st strand DNA|purified 1<sup>st</sup> strand DNA]] | ||
* 2 μL NE Buffer 4 | * 2 μL NE Buffer 4 | ||
* 2 μL cobalt chloride (solution provided with TdT enzyme) | * 2 μL cobalt chloride (solution provided with TdT enzyme) | ||
* 1 μL 2 mM dATP (98 μL ddH<sub>2</sub>O + 2 μL dATP) | * 1 μL 2 mM dATP (98 μL ddH<sub>2</sub>O + 2 μL dATP) | ||
* 1 μL TdT (in freezer; keep on ice) | * 1 μL TdT (in freezer; keep on ice) | ||
===Procedure=== | ===Procedure=== | ||
* Combine | * Combine | ||
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===Round One=== | ===Round One=== | ||
* Use 48°C annealing temperature | * Use 48°C annealing temperature | ||
* Use poly-T primer ([[Griffitts:5'RACE#oJG636|oJG636]]) and outside gene-specific primer | * Use poly-T primer ([[Griffitts:5' RACE#oJG636|oJG636]]) and outside gene-specific primer | ||
===Round Two=== | ===Round Two=== | ||
* Use 54°C annealing temperature | * Use 54°C annealing temperature | ||
* Use nested primer and poly-T primer ([[Griffitts:5'RACE#oJG636|oJG636]]) | * Use nested primer and poly-T primer ([[Griffitts:5' RACE#oJG636|oJG636]]) | ||
* Use template from [[Griffitts:5'RACE#Round One|1st round of PCR]] | * Use template from [[Griffitts:5' RACE#Round One|1st round of PCR]] | ||
Note: A third round with yet another nested primer may be necessarybr> | Note: A third round with yet another nested primer may be necessarybr> | ||
Latest revision as of 11:30, 11 April 2008
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.