restriction of pbluescript - restriction of pbluescript (Jul/08/2005 )

i am working on the pbluescript (2.9kb). i transform it into the E.Coli, and when i isolated i got only one band of 2.3 kb.
i dont know where i was wrong.

what can be the possible reason of disappearance of the pbluescript from the gel when it is restricted digested with the Sma1,

and now i have to synthesise the cdna library.
my approach is that i am having the total cdna(blunt end), and i want to clone the cdna of 1kb (appropriately), so i am taking the molar ratio of 1:3(vector :insert), what possible precaution should i take.

upto what time one cdna can be stored. and how the precipitaton of cdna is done, if any other information related with cdna, then plz tell, i will be highly obliged.

one thing more when i restrict my vector and then did phenol chloroform and after precipitation with .2 M K-acetate overnight , after spin i found no pellet, this prob i generally i faced. if u have any suggestion then plz tell me.



thanx in advance

I don't think you can precipitate DNA with just potassium acetate. Normally, you would separate the supernatant, add 1/10 volume of 3M sodium acetate pH 5.2, then 2.5 volumes of 100% ethanol, chill, and centrifuge.

Thanx for your kind information,


now i have one query that the sodium acetate u told me is for any vector or only for p bluescript special, and after adding abs alcohol for, how much time should i kept it for incubation and at which temp.


thanx

Many people place their plasmid in the -20 or -80 for times ranging from 30 min to overnight. I add my Na Acetate, ethanol and spin.

*pBluescript, who also has the tendancy to precipitate in the presence of alcohol.*

Check out the messages in "Pinned: Tips on ethanol precipitation of nucleic acids and wash" in the General Lab Techniques forum.