Technique / Molecular Biology / Cloning library construction and screening
Cloning long DNA fragments inserts
Cloning long DNA fragments inserts
I had attempted to clone a 5kb insert (from a genomic library) into
the bluescript vector atleast 3 times (into Kpn I - Sma I sites) with
nosuccess. I am doing this only to sequence my inserts. I do a blue
white screening, none of my white colonies contain the insert. One
of my colleagues suggested using the pUC 19 vector.
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Last update 14-Jan-2002, Rating of 0 votes.
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By Jon Rees
on 14-Jan-2002
I agree that cloning the 5kb insert into a vector in both directions might be a safe bet. Alternatively, are you cutting with SmaI at 25 degrees Celsius in one buffer for 1 hour, then purifying, and cutting in another buffer with KpnI at 37? According the NEB catalog, the buffer requirements for the 2 enzymes are different. I might be tempted to try this out. |
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By Jon Rees
on 14-Jan-2002
I agree that cloning the 5kb insert into a vector in both directions might be a safe bet. Alternatively, are you cutting with SmaI at 25 degrees Celsius in one buffer for 1 hour, then purifying, and cutting in another buffer with KpnI at 37? According the NEB catalog, the buffer requirements for the 2 enzymes are different. I might be tempted to try this out. |
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