915
edits
Changes
Rewrote protcol for first strand synthesis
[[ Category: Cloning ]]
==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
===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===
==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
===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===
