View source for 5' RACE

[[ Category: Cloning ]]

__NOTOC__

==Materials==
* 5' RACE Kit (Version 2.0 from Invitrogen; http://products.invitrogen.com/ivgn/product/18374058)
* Double distilled water
* Absolute ethanol
* Total RNA, 1-5 ug
* Gene Specific Primers (see [[ Designing Primers for 5' RACE ]])

==Protocol==
Copied from the manufacturer's protcol [http://tools.invitrogen.com/content/sfs/manuals/5prime_race_man.pdf 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===
<ol>
<li> Combine the following in a PCR tube:</li>

{| border="1"
|-
! Component !! Amount 
|-
| GSP1 || 2.5 pmoles (2.5 uL of 1 uM) 
|-
| RNA || 1-5 ug (from nanodrop)
|-
| DEPC Water || To 15.5 uL
|}

<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> Add the following in order:</li>
# 2.5 uL 10X PCR Buffer 
# 2.5 uL 25mM MgCl2
# 1 uL 10 mM dNTP
# 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> Add 1 uL SuperScript II RT</li>
<li> Incubate 50 min at 42C</li>
<li> Incubate 15min at 70C</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>
</ol>

===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
|-
| 94C || 90s
|-
| 94C || 90s || Repeat 35X
|-
| 55C || 90s || Repeat 35X
|-
| 72C || 2 min || Repeat 35X
|-
| 72C || 7 min
|-
| 4C || Hold
|}
<li>Analyse 5-20uL by agarose gel electrophoresis</li>
</ol>