6 replies to this topic

[sssss]

hiiiii,I hav a query...........
           what will happen if I skip chloroform step during plasmid isolation. After adding equal vol. of phenol chloroform to lysate I centrifuged at high speed.then i directly started th abs. alcohol step.......without goin for chloroform step.
will ther b any problem to plasmid.........After Isolaton I hav 2 go for transformation using that plasmid.......
plssss help

[mastermi]

Usually you do the chloroform step to get rid of the phenol. So maybe you will have some phenol in your plasmid solution.
I have no idea if the transformation does work if there is phenol in he solution. So maybe it would be better to do a chloroform extraction and make a new alcohol precipitation...

[Curtis]

why don't you have them mixed?

I use a mixed solution of 25:24:1 of phenol:chloroform:IAA

[phage434]

There is probably phenol contamination in your preparation, which will likely inhibit anything else you'd like to do.  You might be able to transform, but I would doubt that most enzyme reactions would work.  You can purify your product by extracting with chloroform and re-precipitating.  It might make things easier to dilute the DNA in TE to increase the volume some.

[sssss]

phage434, on Apr 11 2009, 07:29 AM, said:

There is probably phenol contamination in your preparation, which will likely inhibit anything else you'd like to do.  You might be able to transform, but I would doubt that most enzyme reactions would work.  You can purify your product by extracting with chloroform and re-precipitating.  It might make things easier to dilute the DNA in TE to increase the volume some.

thanks a lot..........
actually after digesting the bands hav come on proper size...transformation has also worked fine

[hanming86]

sssss, on Apr 13 2009, 03:25 AM, said:

phage434, on Apr 11 2009, 07:29 AM, said:

There is probably phenol contamination in your preparation, which will likely inhibit anything else you'd like to do.  You might be able to transform, but I would doubt that most enzyme reactions would work.  You can purify your product by extracting with chloroform and re-precipitating.  It might make things easier to dilute the DNA in TE to increase the volume some.

thanks a lot..........
actually after digesting the bands hav come on proper size...transformation has also worked fine

thanks to the 70% ethanol wash which followed.

[sssss]

hanming86, on Apr 13 2009, 05:13 AM, said:

sssss, on Apr 13 2009, 03:25 AM, said:

phage434, on Apr 11 2009, 07:29 AM, said:

There is probably phenol contamination in your preparation, which will likely inhibit anything else you'd like to do.  You might be able to transform, but I would doubt that most enzyme reactions would work.  You can purify your product by extracting with chloroform and re-precipitating.  It might make things easier to dilute the DNA in TE to increase the volume some.

thanks a lot..........
actually after digesting the bands hav come on proper size...transformation has also worked fine

thanks to the 70% ethanol wash which followed.

yaaaaa,
its possible..........