5' RACE: Difference between revisions

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

* Place binding solution at room temperature.

* Warm some sterile water to 65C

===First Strand Synthesis===

Line 47:

Line 49:

===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.

@@ 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
+* Place binding solution at room temperature.
+* Warm some sterile water to 65C
 ===First Strand Synthesis===
@@ Line 47: / Line 49: @@
 ===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.