5' RACE - Revision history

Davebrid: /* PRC of dC-Tailed cDNA */

2012-12-04T21:04:24Z

PRC of dC-Tailed cDNA

← Older revision		Revision as of 21:04, 4 December 2012
Line 73:		Line 73:
	# Heat inactivate at 65C for 10min.		# Heat inactivate at 65C for 10min.

	===~~PRC~~ of dC-Tailed cDNA===		===PCR of dC-Tailed cDNA===
	<ol>		<ol>
	<li> Prepare a master mix containing the following (multiply by the number of samples) and aliquot 44.5 uL into PCR tubes.</li>		<li> Prepare a master mix containing the following (multiply by the number of samples) and aliquot 44.5 uL into PCR tubes.</li>

Davebrid: Added PCR reaction

2012-12-04T21:04:12Z

Added PCR reaction

@@ Line 1: / Line 1: @@
 [[ Category: Cloning ]]
 ==Materials==
@@ Line 33: / 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> Add the following in order:</li>
@@ Line 40: / Line 42: @@
 # 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> 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>
+<li> Incubate 30min at 37C using the PCR Program '''5 RACE RNAse H'''.  Can freeze at -20 or continue to cDNA Purification</li>
 </ol>
@@ Line 67: / Line 69: @@
 ## 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'''.
+# 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.

Davebrid: Added purification and tailing protocols

2012-12-04T14:49:27Z

Added purification and tailing protocols

@@ Line 15: / Line 15: @@
 * 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===
@@ Line 47: / Line 49: @@
 ===cDNA Purification===

Davebrid: Rewrote protcol for first strand synthesis

2012-12-04T14:36:09Z

Rewrote protcol for first strand synthesis

New page

[[ 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===