915
edits
Changes
Added purification and tailing protocols
* 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===
===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.