5' RACE: Difference between revisions
Added purification and tailing protocols |
Added PCR reaction |
||
| Line 1: | Line 1: | ||
[[ Category: Cloning ]] | [[ Category: Cloning ]] | ||
__NOTOC__ | |||
==Materials== | ==Materials== | ||
| Line 33: | Line 35: | ||
|} | |} | ||
<li> Incubate at 70C for 10 min to denature RNA using the PCR Program '''5 | <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 40: | 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 | <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 | <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> | ||
| Line 67: | Line 69: | ||
## 2.5uL 2mM dCTP. | ## 2.5uL 2mM dCTP. | ||
## 10 uL Purified cDNA. | ## 10 uL Purified cDNA. | ||
# Incubate at 94C for 2-3 min. This and the next two steps are in the PCR program '''5 | # 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. | # Add 1 uL TdT, mix and incubate 10 min at 37C. | ||
# Heat inactivate at 65C for 10min. | # 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> | |||