End-labeling DNA fragments
End-labeled DNA fragments are used in a wide variety of
molecular biology experiments. Doubley end-labeled fragments are useful
as MW standards on Southern blots, as probes for filter-binding or gel
retardation experiments, or for visualizing small amounts of restricted
DNA. Singley end-labeled DNA is used for Maxam & Gilbert sequencing,
for S1 analysis, footprinting, primer extension, etc.
This method can also be used for 5' end-labeling RNA molecules.
10X Kinase buffer Prep Gel
------------------ ----------------
0.5M Tris,pH9.5 48.3ml ddH2O
0.1M MgCl2 25ml glycerol
50mM DTT 60mg APS
50% glycerol 16.6ml 29%:1% acrylamide:bis
10ml 10X TBE
Denaturation buffer
--------------------
1M Tris, pH9.5 Elution buffer
10mM Spermidine ----------------
1mM EDTA 0.5M NH4OAc
10mM Mg(OAc)2
Phenol:chloroform 1mM EDTA
------------------- 0.1% SDS
48% chloroform
50% TE sat'd phenol
2% isoamyl alcohol
1 - Digest 10ug DNA with the appropriate restriction enzme (the one you
want to label at) overnight in a 30ul reaction. If you desire or need
to, a larger reaction can be used, then the DNA should be EtOH
precipitated and resuspended in 30ul 1X restriction buffer.
2 - Add 3.5ul 1M Tris, pH8, and 2ul 4u/ul CIAP (calf intestine alkaline
phosphatase). Incubate 30 min at 56C.
3 - Add 56ul 2M NH4OAc and 156ul ddH2O. Add 200ul phenol:chloroform,
vortex VIGOROUSLY, then spin 1 min. Collect the upper (aqueous) phase
and again add 200ul phenol:chloroform, vortex, and spin as before.
Collect the upper phase, and add 600ul EtOH and freeze for 5 min in a
dry-ice:EtOH bath. Spin for 5 min, discard the supernatant, and add
750ul ice-cold 70% EtOH. Again freeze for 5 min in a dry-ice:EtOH bath,
spin for 5 min, and discard the supernatant. Dry the pellet in a
rotovac.