ࡱ> .0-c  bjbj** .HSbHSbT]44444HHH8HF($>d44444PTa;R0FXX"X4EFXB : Oligonucleotide 5 adenylation Using a 5 phosphorylated oligonucleotide and the 5 adenylation kit from NEB Set up reaction: Phosphorylated DNA Oligonucleotide 5 l (500 pmol) 10x 5 DNA Adenylation Reaction Buffer 5 l 1 mM ATP 5 l Nuclease-free Water to 45 l Mth RNA Ligase 5 l Incubate at 65C for 1 hour. Incubate 85(C for 5min Lid at 95deg Dilute to 100ul with water, extract with 100l phenol:chloroform pH8 Add 10l 3M NaOAc, 1l glycogen and 250l 100% ethanol Vortex Spin 15 min top speed at 4(C Wash pellet with 400l 70% ethanol Normal oligos use this for TrAEL-seq adaptor Dissolve pellet in 50ul 0.1xTE (to 10pM/l final concentration) Vortex then let sit at room temperature for 30min or so Store at -30(C Hairpin adaptors Dissolve pellet in 45ul 0.1xTE (to 10pM/l final concentration) Vortex then let sit at room temperature for a few minutes Add 5l 10x buffer Use T4 DNA ligase buffer for ENDseq, NEBuffer 1 for LIG-seq Place in a heating block at 95(C, remove block from the heat and allow to cool to room temperature over a few hours Store at -30(C      FILENAME Oligo adenylation and purification v1.0 Houseley lab  PAGE 1  S T }  5 B C c d e   P B C D P Q S T U W X Z [ ] ^ ` ¾ޯdzhTjhTU h^TZh^TZ jhQh^TZ jh^TZhC0 hQh^TZh)h^TZ htfh^TZhtf htf6 h^TZ6hmh1x jhQhQ hQhQhMh)5hM5CJ(aJ(hQ5CJ(aJ(/ no ) * G H _ m n   4 `gdm`gdQgdQgd)$a$gd)4 5 d e  A | } C D S T V W Y Z \ ] _ ` gd^TZgdQ` a k l h^TZh^TZhThpv)0JmHnHu h(0Jjh(0JUh%hpv)mHnHuh(jh(U` gdQ,1h. A!"#$% s2 0@P`p2( 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p 0@P`p8XV~ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@ 0@_HmH nH sH tH @`@ NormalCJ_HaJmH sH tH DA`D Default Paragraph FontRiR  Table Normal4 l4a (k (No List 4@4 Mr2Header  9r 4 @4 Mr2Footer  9r .)@. Mr2 Page NumberPK![Content_Types].xmlN0EH-J@%ǎǢ|ș$زULTB l,3;rØJB+$G]7O٭Vc:E3v@P~Ds |w< " OO\\\_`  4 `  :PWY_!8@0(  B S  ?<BTVWYZ\]_`TVWYZ\]_`3} 35tSTTWW`} 35tSF