Genomic Library Screening

futers at bpxtal.leeds.ac.uk futers at bpxtal.leeds.ac.uk
Mon Feb 28 06:55:08 EST 1994


In article <1994Feb26.213031.1903824 at sue.cc.uregina.ca>,
    ZACHARIAS at Meena.CC.URegina.CA (Tracey Zacharias) writes:
>I am screening a lambda genomic library and think I have some positives. 	
>I have picked them, grown them up in LB + E.coli and now want to screen
>them again.  I am thinking of just taking about 5 microL and dotting 
>it on a membrane and then treating just as if I had done a plaque 
>lift.  Is this a reasonable approach?  I cannot find any references 
>that do it this way.  Does the LB that the lambda was grown up in have
>an effect? Will I get a bunch of positives that aren't really positive 
>
Why have you grown the positives after picking the plaques off the initial 
screening plates?  The plaque you have picked will contain the putative 
positive clone mixed with clones from surrounding phage.  Therefore,
rescreening by applying a dot of the regrown culture will only show which
isolates contain the positive you are after.  The strength of the signal will
depend on how well the positive page will grow in relation to the contaminating
phage.  You will still have to purify the positive afterwards as well as the
possibility that you will lose it due to a faster growing contaminating phage.
I suggest you pick the positive plaque and replate at a lower density than the
initial screening.  Repeat this rescreening procedure until every plaque
on the plate is positive and hence the phage stock is pure.
I hope this is helpful.
              Simon Futers   (futers at biovax.leeds.ac.uk) 
>Zacharit at max.cc.uregina.ca 



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