cloning problem

Marek Nelke MNELKE at IMAIL.NSAC.NS.CA
Thu Apr 16 15:54:36 EST 1998


Hi!

I isolated  clone from red clover lambda genomic library (in EMBL3) 
wich strongly reacts with the cDNA probe from soybean. I isolated the 
DNA insert (~ 12kb) by Sfi I digestion (the insert hybridizes to the 
cDNA probe as shown by Southern). When this 12 kb fragment was 
digested with Pal I it generated 3.4 kb band which exclusively reacts 
with the soybean cDNA. Restriction mapping of 12 kb fragment 
showed that  3.4 kb fragment does not have Sfi I border. I "gene 
cleaned" this 3.4 kb fragment and ligated to dephosphorylated Sma I 
pUC18. After transformation I got some white colonies, but after 
plasmid purification and sequencing all of the "positive clones" they 
occured to be part of pUC18 sequence. When the pUC18s, which supposed 
to carry the red clover DNA insert, were digested with  restriction 
enzymes (Sal I and Ban II) which preceed Sma I cloning site, I got on 
gel 2.8 kb band ( whole plasmid) and additional band (1.3 - 1.6 kb, 
plasmid fragments) for each tested plasmid.  For one of my ligation 
 I used somebody else reagents (except for pUC), who used them to 
sussessfully clone different DNA fragment. It also did not work for 
me. Has somebody else had similar clonning problem? Any 
suggestions how to overcome this cloning problem are welcome.


Marek Nelke
MNelke at Imail.nsac.ns.ca
Nova Scotia Agricultural College
Plant Science
PO  Box 550
Truro, Nova Scotia B2N 5E3
Canada





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