Query about phage

SPIC BIOPROCESS biospic at GIASMD01.VSNL.NET.IN
Tue Jul 9 10:16:32 EST 1996


From

P. Lalitha
Centre for Biotechnology
Anna University
Madras 600 025
Tel 91 44 2350298
Fax 91 44 2350299
email : biospic at giasmd01.vsnl.net.in


I   am  working  with  the  cDNA  library  of  W.  bancrofti
constructed  in  lambda Zap II vector. While  screening  the
library   with   filarial  antibodies   we  came  across   a
particular  phage  which lysis the E.coli  strain  XL1  Blue
within four hours. The titre of  the phage is extremely high
(around  10exp12).  Since this particular phage reacts very
strongly  with  the antisera  we tried to characterise  this
phage.

USING VARIOUS STRAINS OF E.COLI:
With KL-226 it does not form plaques. This confirms that  it
is not a T1 phage.
With  Q358 and LE392 strain  it forms plaques this  confirms
it is a lambda phage.
With  CSH57() strain it doesn't form plaques. This  confirms
that it is a Lytic lambda.

DNA EXPERIMENTS:
   The  phage  DNA was extracted according to the prescribed
protocols as in Mannitias et al 1989. The size of the  Phage
DNA  was confirmed by running it in a 0.5% agarose gel using
the  wild  type lambda and the DNA marker. Next we tried  to
see  the  restriction pattern of the DNA using the Hind  III
enzyme  for  linearisation and Xho1/Xba1 for double  digest.
But  we did not see any restriction pattern and only streaks
were visible.

CONVERSATION INTO PLASMID:
Since  the titre of the Phage is very high we were not  able
to convert it into plasmid using helper phage .

I NEED THE FOLLOWING HELP

1.   Why  streaks  appear  when  digested  with  restriction
enzymes?.

2.  Why this phage lysis within  4hrs and what is the reason
for very high titre?

Can any one help me regarding this phage.






More information about the Methods mailing list