cloning

Jarmo Niemi TUY JANIEMI at FINABO.ABO.FI
Thu Nov 26 06:12:50 EST 1992


In <01GRKOX8SIFK001160 at uvmvax.uvm.edu> W_SHAO at UVMVAX.BITNET writes:

> Hi, there:
> I am trying to clone a gene from lambda gt11 expression library with a
> oligonucleotide probe. Usually, one has to make a set of duplicates of
> nitrocellulose filter to confirm the spots are real. I heard that
> duplicates are not really reliable. Is this true?
> Thanks for response.
> W Shao

Reliable in what sense ? We screened a lambda EMBL4 bank using a weak
cross-hybridisation (random-primed probe) and the use of duplicates seemed
to be essential, otherwise one would end up cloning several spurious spots,
which gave no signal on rescreening. I think all clones, that were positive
on duplicate filters, were positive on rescreening. In my experience one 
can pick up to 5 duplicate filters from a plate; the last one gives already
a weakened signal. We used Colony-Plaque Screen from NEN.

Jarmo Niemi   Biochemistry, University of Turku
              SF-20500 Turku, Finland
              ++358-21-6335758
              janiemi at finabo.abo.fi

"You may quote me"
	-this sentence
		(quoted from Douglas R. Hofstadter:Metamagical themas)
			{therein reprinted from Scientific American,
			January, 1981}
				[and probably published even before]



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