S1 Oligo Gel Purification
Kinase one oligo in a 25ul reaction:
Crude dephosphorylated oligo 0.5 pmol
10x T4 PNK buffer 2.5μl
gamma-32P-ATP(specific activity > 7000Ci/mmol) 11.9pmol
T4 PNK (10U/ul) (NEB) 10U
--Incubate at 37ºC for 1hr.
--Mix 2000pmol of cold crude oligo (500pmol/ul) with 5μl of above kinase
reaction and 100% formamide dye for a final concentration of 60% of dye
--Heat 100ºC for 3 min. and ice for ~10 min., load on to a 0.8mm thick 8%
acrylamide (19:1), 8M Urea, 1x TBE denaturing gel with the widest comb, run
2 hr.15 min at 550 Volts with fan (until BPB is at the bottom of gel)
--Separate plates and wrap gel + larger glass plate in saran wrap
--With the film on the counter and the gel sandwiched between the film and the
glass plate, expose for 30 min. to gray film using the safety light with its
shutters mostly closed to expose the outline of the plate and gel. (Make sure
exposing gel is shielded) Then develop.
--Using a light box, line up film with gel to locate labeled oligo, cut out band and
transfer to 350μl of elution buffer.
--Crush band and elute overnight at 37ºC
--Vortex elution mixture, quick spin, transfer to fresh tube, repeat 1-2x
(Want no acrylamide in end sample)
--Add 1ml iced EtOH, precip. 1 hr at –70ºC, spin 15 min in radioactive
microcentifuge at 4C, wash 2x with 70% EtOH, dry, resuspend in
50μl TE, pH 8.0
Quanitations:
--Dilute 1μl of above kinase rxn 1:100 in 0.2M EDTA, mix, spot 3μl of dilution
on a small nitrocellulose filter, allow to dry (Total counts)
--Take 3μl of diluted kinase rxn and add to 100μl of 0.1mg/ml BSA + 20mM
EDTA, mix
--To above mixture, add 1.3mL iced 10% TCA 1% sodiumpyrophosphate, mix,
ice for 20 min.
--Vacuum filter the precipitate onto a nitrocellulose filter, wash 3x with 5mL iced
10% TCA, wash 1x with 95% EtOH, allow to dry (Incorporated counts)
--Spot 2.5μl of the 50μl eluted oligo onto small nitrocellulose filter, allow to dry
(Band Counts)
--Use a Geiger Counter to read the cpm of the three filters, keeping the detector at
the same distance from each filter and recording the average reading
Calculations:
% counts incorp. = [(incorp. counts)/ (total counts)] x 100
% oligo labeled = {[(fraction of total counts incorp.) x ( 11.9 pmol gamma ATP)] / 0.5 pmol oligo} x 100
cpm of labeled oligo added to gel = [(incorp counts / 3μl) x 100 dilution factor x 5μl]
% labeled oligo that is right size = {[(Band counts/2.5μl) x 50μl] / (cpm of labeled oligo added to gel)} x 100
Concentration of eluted oligo = [2000pmol x (fraction of labeled oligo that is the right size)] / 50μl
Dilute to 1pmol per μl with TE, pH 8.0
Elution Buffer:
0.5M NH2OAc
1mM EDTA
0.1% SDS