915
edits
Changes
Added RNA tailing
[[ Category: Transcription ]]
__NOTOC__
see [[ SOP - Chloroform ]] for safety information about working with Chloroform.
This is adapted from [http://dx.doi.org Yang ''et al'' 2010]
==Materials==
* Purify total RNA, via the protocol: [[ Purification of miRNA and mRNA with TRIzol ]]
* PolyA polymerase (NEB cat# M0276S)
* Phenol/Choroform mixture (1:1 ratio)
* 3M Sodium acetate pH 5.2
* Isopropanol
* Superscript III reverse transcriptase (Life Technologies Catalog # 18080093)
* Oligo dT Adapter Primer '''5'GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3''', dissolved to 50 uM
==Protocol==
===PolyA Tailing of RNA===
* Incubate at 37°C for 1 hour. Components include
** 1 ug RNA
** 2 uL 10X polymerase reaction buffer
** 2 uL ATP (10 mM, comes with polymerase)
** 1 uL poly(A) polymerase
** ddH2O to a final volume of 20 uL
* Add 160 uL Water, 20 uL of 3 M sodium acetate, pH 5.2 and 200 uL of phenol/chloroform to tailed RNA, mix by tapping to form an emulsion
* Centrifuge for 1 min on max to separate layers
* Transfer the upper (aqueous) layer to a clean tube. Add 200 uL chloroform and mix, spin and extract aqueous layer to a new tube twice to remove the excess phenol
* Add 140 uL of isopropanol, mix and incubate 5 minutes. Centrifuge 15 minutes on max. Aspirate supernatant being careful not to touch the pellet and air dry for 5-10 mins
* Reedissolved in 25 μL of water
===Reverse Transcriptase===
** 1 uL of Oligo dT Primer
** 1uL of dNTP
** 500 ng 6uL of tailed RNA** 5 uL Sterile water up to 13 uL
* Heat at 65C for 5 minutes
* Briefly centrifugre then add (per reaction):