cloning-lambda-inserts

Lauri Lintott llintott at ACS.UCALGARY.CA
Fri Oct 28 14:58:51 EST 1994


 I have isolated several lambda clones from a Drosophila EMBL4 
genomic library using a cDNA probe.  Upon digestion of the 
isolated lambda DNA with EcoRI two clones release several smaller 
fragments (8 kbp to 700 bp) from the insert.  Three clones each released 
one fragment of about 15 kbp from the insert.  I subcloned these 
fragments into pBluescript SK+ and sequenced their ends.  A BLAST 
search using obtained sequences indicates that the 
smaller fragments are Drosophila DNA but the 15 
kbp fragments are E. coli DNA.  Furthermore I have been unable to 
clone the 8 kbp fragment from one of the multiple EcoRI fragment 
clones.  Obviously during my lambda DNA isolation E. coli DNA 
released during lysis of the host is not being completely degraded.  
I have tried both Sigma DNAse I, type IV from bovine pancreas and 
Pharmacia DNAse I.  The presence of contaminating E. coli DNA has 
apparently not affected my ability to successfully clone the smaller 
pieces.  Presumably because the desired pieces are in great excess 
to the contaminating E. coli DNA.  This should be true for the 15 
kbp pieces as well.  Several question arise from this:
1.  Can anyone suggest a better DNAse I to use for this procedure 
(essentially that for small scale lambda DNA preparation from 
Current Protocols)?
2.  Has anyone ever experienced an inability to clone large pieces 
of genomic library DNA into pBluescript using E. coli DH10B, DH5a or 
XL-1 Blue (I have tried each of these hosts for the 8 kbp piece and 
one of the 15 kbp pieces)?  Is this because large pieces of foreign 
DNA are toxic to E. coli but large E. coli pieces are not?  Will 
cutting these large pieces up using other enzymes help with their 
recovery?

Any suggestions are welcome.  Thanks.









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