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created page and initial set up
For labeling cDNA probes for use for [[In Situ Hybridization]].
'''Reagents:'''
High Prime DNA Labeling Kit, 50 reactions, Roche Applied Science, #11585584001 $333
dATP-alphaS35, (250 uCi vial = 20 µl) Perkin Elmer #NEG034H250UC $172
:made first Tuesday of every month
Spin columns.
'''Procedure:'''
Take radioactivity out to melt (1 h if isotope in thick glass vial).
Turn on heat block to 100° C.
In 1.5 ml tube:
*100 ng cDNA probe
*1 µl DTT 250 mM stock solution
*ddH20 to make 8 µl
Denature at 100 C for 10 min (put cap lock on tube).
Cool on ice 2-3 minutes, spin down
Add:
*1 µl each dCTP, dGTP, dTTP (tubes 3,4,5)
*4 µl of 5x Klenows enzyme/hexanucleotide mixture (tube 6)
*5 µl 35S-dATP
(20 µl total)
Spin down. Leave on bench at RT for 12-24 h. (or at 37C for 1 h).
Add 30µl TE to stop reaction.
Spin down in spin column.
Count 1 µl in 10 ml scintillation fluid.
Should be 500,000 to 2,000,000 cpm/µl
If more than 1,000,000 cpm, there may be excess unincorporated nucleotides that can be removed with a second spin column run.
Use 5-10 million cpm / ml of hybridization buffer for in situ hybridization.
'''Reagents:'''
High Prime DNA Labeling Kit, 50 reactions, Roche Applied Science, #11585584001 $333
dATP-alphaS35, (250 uCi vial = 20 µl) Perkin Elmer #NEG034H250UC $172
:made first Tuesday of every month
Spin columns.
'''Procedure:'''
Take radioactivity out to melt (1 h if isotope in thick glass vial).
Turn on heat block to 100° C.
In 1.5 ml tube:
*100 ng cDNA probe
*1 µl DTT 250 mM stock solution
*ddH20 to make 8 µl
Denature at 100 C for 10 min (put cap lock on tube).
Cool on ice 2-3 minutes, spin down
Add:
*1 µl each dCTP, dGTP, dTTP (tubes 3,4,5)
*4 µl of 5x Klenows enzyme/hexanucleotide mixture (tube 6)
*5 µl 35S-dATP
(20 µl total)
Spin down. Leave on bench at RT for 12-24 h. (or at 37C for 1 h).
Add 30µl TE to stop reaction.
Spin down in spin column.
Count 1 µl in 10 ml scintillation fluid.
Should be 500,000 to 2,000,000 cpm/µl
If more than 1,000,000 cpm, there may be excess unincorporated nucleotides that can be removed with a second spin column run.
Use 5-10 million cpm / ml of hybridization buffer for in situ hybridization.