single stranded DNA prep

[Malathi Hari]

 I am trying to prepare single stranded DNA from a construct in pBS for
site directed mutagenesis. After a few tries I am still getting a lot of of
ds plasmid DNA in my prep, as deduced by the ability of the ssDNA to transform
bacteria and digestion of the DNA;eading to fewr number of transformants.
I was wondering if anybody out there would have soem ideas.
In brief this is what I do :
Grow up an o/n
Inoculate 1:100 and grow up for 1 hour (I have tried 1.5 ml and 15 ml volumes)
Add R408 rescue phage ( I don't exactly know the pfu, but I do get lysis as
seen by the appearance of coagulum)
Spin down and to sup add PEG/NaCl and keep on ice 30 min to 2hrs
Spin at high speed
Dissolve pellet in TE
Phenol extract
Chloroform extract
Ethanol ppt
70% wash

ANY IDEAS???

Malathi


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Malathi Hari					(Home)
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