Difference between revisions of "5' RACE"

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(Rewrote protcol for first strand synthesis)
 
(→‎PRC of dC-Tailed cDNA)
 
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[[ Category: Cloning ]]
 
[[ Category: Cloning ]]
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__NOTOC__
  
 
==Materials==
 
==Materials==
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* Prepare and chill wash buffer by adding 18 mL water, 21 mL ethanol and 1 mL wash buffer concentrate into a glass bottle.  Place at 4C
 
* Prepare and chill wash buffer by adding 18 mL water, 21 mL ethanol and 1 mL wash buffer concentrate into a glass bottle.  Place at 4C
 
* Prepare and chill 70% ethanol by adding 35 mL ethanol to 15 mL water in a glass bottle.  Place at 4C
 
* Prepare and chill 70% ethanol by adding 35 mL ethanol to 15 mL water in a glass bottle.  Place at 4C
 +
* Place binding solution at room temperature.
 +
* Warm some sterile water to 65C
  
 
===First Strand Synthesis===
 
===First Strand Synthesis===
Line 31: Line 35:
 
|}
 
|}
  
−
<li> Incubate at 70C for 10 min to denature RNA using the PCR Program '''5' RACE 70C'''</li>
+
<li> Incubate at 70C for 10 min to denature RNA using the PCR Program '''5 RACE 70C'''</li>
 
<li> Place on ice for 1 min</li>
 
<li> Place on ice for 1 min</li>
 
<li> Add the following in order:</li>
 
<li> Add the following in order:</li>
Line 38: Line 42:
 
# 1 uL 10 mM dNTP
 
# 1 uL 10 mM dNTP
 
# 2.5 uL DTT
 
# 2.5 uL DTT
−
<li> Incubate 1 min at 42C using the PCR program '''5' RACE First Strand''', which covers the next three steps</li>
+
<li> Incubate 1 min at 42C using the PCR program '''5 RACE First Strand''', which covers the next three steps</li>
 
<li> Add 1 uL SuperScript II RT</li>
 
<li> Add 1 uL SuperScript II RT</li>
 
<li> Incubate 50 min at 42C</li>
 
<li> Incubate 50 min at 42C</li>
 
<li> Incubate 15min at 70C</li>
 
<li> Incubate 15min at 70C</li>
 
<li> Remove and add 1uL RNAse H, mix thoroughly</li>
 
<li> Remove and add 1uL RNAse H, mix thoroughly</li>
−
<li> Incubate 30min at 37c using the PCR Program '''5' RACE RNAse H'''.  Can freeze at -20 or continue to cDNA Purification</li>
+
<li> Incubate 30min at 37C using the PCR Program '''5 RACE RNAse H'''.  Can freeze at -20 or continue to cDNA Purification</li>
 
</ol>
 
</ol>
  
 
===cDNA Purification===
 
===cDNA Purification===
 +
# Add 120 uL binding solution to the first strand reaction.
 +
# Transfer reaction to a SNAP column provided.
 +
# Spin at 13K for 20s.
 +
# Remove the cartridge and transfer flow through to a new microcentrifuge tube.  Save until cDNA recovery is confirmed.
 +
# Add 400 uL cold wash buffer to the spin cartridge, replaced in microcentrifuge tube.
 +
# Spin at 13K for 20s.
 +
# Repeat wash '''three''' more times.
 +
# Wash the cartridge twice with 400 uL cold 70C
 +
# After second wash, centrifuge 1 min at 13K to removed residual ethanol.
 +
# Transfer cartridge into a clean recovery tube.  Add 50 uL of preheated water.
 +
# Spin at 13K for 20s.
 +
 +
===TdT Tailing of cDNA===
 +
# To a new tube add the following:
 +
## 6.5uL DEPC Treated water.
 +
## 5uL 5X tailing buffer.
 +
## 2.5uL 2mM dCTP.
 +
## 10 uL Purified cDNA.
 +
# Incubate at 94C for 2-3 min.  This and the next two steps are in the PCR program '''5 RACE TdT'''.
 +
# Add 1 uL TdT, mix and incubate 10 min at 37C.
 +
# Heat inactivate at 65C for 10min.
 +
 +
===PCR of dC-Tailed cDNA===
 +
<ol>
 +
<li> Prepare a master mix containing the following (multiply by the number of samples) and aliquot 44.5 uL into PCR tubes.</li>
 +
{| border="1"
 +
|-
 +
! Component !! Amount
 +
|-
 +
| Water || 31.5 uL
 +
|-
 +
| 10X PCR Buffer || 5 uL
 +
|-
 +
| 25 mM MgCl2 || 3 uL
 +
|-
 +
| 10 mM dNTP || 1 uL
 +
|-
 +
| GSP2 || 2 uL (of a 10 uM solution)
 +
|-
 +
| Abridged Anchor Primer || 2 uL
 +
|}
 +
<li>Add 5 uL of tailed cDNA from previous step.</li>
 +
<li>Add 0.5 uL of Taq immediately prior to mixing.</li>
 +
<li>Transfer to PCR machine running the program '''5 RACE PCR''':</li>
 +
{| border="1"
 +
|-
 +
! Temperature !! Time !! Repeat
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|-
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| 94C || 90s
 +
|-
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| 94C || 90s || Repeat 35X
 +
|-
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| 55C || 90s || Repeat 35X
 +
|-
 +
| 72C || 2 min || Repeat 35X
 +
|-
 +
| 72C || 7 min
 +
|-
 +
| 4C || Hold
 +
|}
 +
<li>Analyse 5-20uL by agarose gel electrophoresis</li>
 +
</ol>

Latest revision as of 21:04, 4 December 2012



Materials

Protocol

Copied from the manufacturer's protcol here

Preparation

  • Resuspend gene specific primers to 1 uM
  • Prepare and chill wash buffer by adding 18 mL water, 21 mL ethanol and 1 mL wash buffer concentrate into a glass bottle. Place at 4C
  • Prepare and chill 70% ethanol by adding 35 mL ethanol to 15 mL water in a glass bottle. Place at 4C
  • Place binding solution at room temperature.
  • Warm some sterile water to 65C

First Strand Synthesis

  1. Combine the following in a PCR tube:
    2. Component Amount
    GSP1 2.5 pmoles (2.5 uL of 1 uM)
    RNA 1-5 ug (from nanodrop)
    DEPC Water To 15.5 uL
  2. Incubate at 70C for 10 min to denature RNA using the PCR Program 5 RACE 70C
  3. Place on ice for 1 min
  4. Add the following in order:
    1. 2.5 uL 10X PCR Buffer
    2. 2.5 uL 25mM MgCl2
    3. 1 uL 10 mM dNTP
    4. 2.5 uL DTT
  5. Incubate 1 min at 42C using the PCR program 5 RACE First Strand, which covers the next three steps
  6. Add 1 uL SuperScript II RT
  7. Incubate 50 min at 42C
  8. Incubate 15min at 70C
  9. Remove and add 1uL RNAse H, mix thoroughly
  10. Incubate 30min at 37C using the PCR Program 5 RACE RNAse H. Can freeze at -20 or continue to cDNA Purification

cDNA Purification

  1. Add 120 uL binding solution to the first strand reaction.
  2. Transfer reaction to a SNAP column provided.
  3. Spin at 13K for 20s.
  4. Remove the cartridge and transfer flow through to a new microcentrifuge tube. Save until cDNA recovery is confirmed.
  5. Add 400 uL cold wash buffer to the spin cartridge, replaced in microcentrifuge tube.
  6. Spin at 13K for 20s.
  7. Repeat wash three more times.
  8. Wash the cartridge twice with 400 uL cold 70C
  9. After second wash, centrifuge 1 min at 13K to removed residual ethanol.
  10. Transfer cartridge into a clean recovery tube. Add 50 uL of preheated water.
  11. Spin at 13K for 20s.

TdT Tailing of cDNA

  1. To a new tube add the following:
    1. 6.5uL DEPC Treated water.
    2. 5uL 5X tailing buffer.
    3. 2.5uL 2mM dCTP.
    4. 10 uL Purified cDNA.
  2. Incubate at 94C for 2-3 min. This and the next two steps are in the PCR program 5 RACE TdT.
  3. Add 1 uL TdT, mix and incubate 10 min at 37C.
  4. Heat inactivate at 65C for 10min.

PCR of dC-Tailed cDNA

  1. Prepare a master mix containing the following (multiply by the number of samples) and aliquot 44.5 uL into PCR tubes.
    2. Component Amount
    Water 31.5 uL
    10X PCR Buffer 5 uL
    25 mM MgCl2 3 uL
    10 mM dNTP 1 uL
    GSP2 2 uL (of a 10 uM solution)
    Abridged Anchor Primer 2 uL
  2. Add 5 uL of tailed cDNA from previous step.
  3. Add 0.5 uL of Taq immediately prior to mixing.
  4. Transfer to PCR machine running the program 5 RACE PCR:
    5. Temperature Time Repeat
    94C 90s
    94C 90s Repeat 35X
    55C 90s Repeat 35X
    72C 2 min Repeat 35X
    72C 7 min
    4C Hold
  5. Analyse 5-20uL by agarose gel electrophoresis
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