Help with a knotty ligation?
TFITZWATER at somalogic.COM
Tue Dec 18 21:39:12 EST 2001
I now strongly suspect that your problem is the CIAP step.
1) The most common cloning problem reported is the inability to add insert
DNA to a BAP- or CIAP-treated vector. Incompletely inactivated phosphatase
in the ligation reaction or exonuclease degradation of the vector termini
from excess phosphatase are the most likely causes. CIAP and BAP are among
the most difficult enzymes to purify and each lot contains a variable amount
of exonuclease contamination.
You must accurately determine the number of ends you wish to
desphosphorylate and use exactly enough CIAP for that quantity of ends. Use
exactly the amount of CIAP recommended by your supplier, as suppliers have
different unit definitions. Caveat: OD260 determinations have a large
margin of error (up to 10-fold). If at all possible, determine the
concentration with a fluorimeter instead.
Because the byproducts of the reaction inhibit the reaction itself
(dephosphorylation generally only proceeds to 95% completion), I then clean
up the DNA by adding 10x stop mix consisting of 200 mM EGTA, pH 8.3, 10x
TNE100 (1x TE containing 100 mM NaCl) and 10% SDS and heat inactivating at
56-68°C for 15-45 minutes; followed by phenol:chloroform extraction and
ethanol precipitation. Then I repeat the entire CIP protocol all over again
to dephosphorylate the residual molecules that failed to dephosphorylate the
first time. This has proven to be infinitely safer than using too much
SAP does not have the exonuclease contamination problem, making life much
simpler. I recommend switching. SAP is completely inactivated by heat.
3) Incubation at 30 degrees in SOC for 90 minutes replaces 37 degrees for 60
minutes following heat pulse. Incubation of the plates at 30 degrees
generally adds only 3 or 4 hours to the time required to get blue/white
color development. Growing the colonies in broth for plasmid preps at 30
degrees is a standard overnight, as the culture reaches the plateau phase
before you get back in the morning. (But, like I said, the CIAP is probably
the culprit here.)
Ligations are performed in 20 uL reaction volumes consisting of 0.5 pmol of
vector and 1 pmol of insert in freshly prepared buffer composed of 33 mM
Tris acetate, pH 7.9, 10 mM magnesium acetate, 66 mM potassium acetate, 100
µg/ml bovine serum albumin (BSA), 0.5 mM DTT and 1 mM ATP. (Many
restriction enzyme suppliers offer a restriction buffer similar to this;
just add ATP.) Add 1 Weiss unit of T4 DNA ligase and incubate the reaction
at 14°C for 3-18 hours. Heat inactivate the ligase at 65°C for 5 minutes
Ligation at 14 degrees overnight is optimal (A. Hu, BRL Focus, 1983, Effect
of Temperature on ligation of Hind III-cleaved DNA 5(3) 13). You can set up
a 14 degree bath in an ice bucket containing a large volume of 14 degree
water and a floating tube rack. Tape the lid down completely around the
circumference with labeling tape and place the bucket in a cold room. The
bath will be about 12 degrees the next morning. Ligation kits generally
have short incubation times but sacrifice total number of transformants.
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