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Susanne Rohrer srohrerNOSPAM at immv.unizh.ch
Wed Jan 19 04:33:07 EST 2000

navita astuti wrote:

> My teacher suggest me to find any procedure of
> detecting DNA insert without digestion with
> restriction enzyme.

Rusconi Mix (That's what we call it, maybe you can find a reference when
searching Medline....)

500ul Lysozyme (10mg/ml)
500ul 0.5M EDTA
250ul 1M Tris pH 7.5
100ul RNase (10mg/ml)
1.15ml Glycerol (87%)
0.02%Bromophenol blue (endconc. of 2mg/10ml)
7.5ml H2O

store at -20 in aliquots

suspend 1 colony in 12-15ul
let stand at RT 10-15 min
add 2ul Phenol, vortex, spin 2'

Load aqueous supernatant on gel - plasmids with insert will  run slower
so you can see it, I hope your insert is big enough...

Good luck!

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