915
edits
Changes
Added PCR reaction
[[ Category: Cloning ]]
__NOTOC__
==Materials==
|}
<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>
# 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 37C using the PCR Program '''5' RACE RNAse H'''. Can freeze at -20 or continue to cDNA Purification</li>
</ol>
## 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.
===PRC 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>
