Miscellaneous Notes About Dephosphorylation of Plasmid DNA

2015-01-13

azim58 - Miscellaneous Notes About Dephosphorylation of Plasmid DNA

Section 1.93 of Molecular Cloning Book (Dephosphorylation of plasmid DNA)

* they recommend digesting with a 2-3 fold excess of the restriction
  enzyme
* they ethanol precipitate before treating with CIP or SAP
* they deactivate the enzyme after use and say that this is crucial.
  They also say that heat inactivation may not be effective enough

This link
http://bitesizebio.com/articles/should-you-use-calf-intestinal-alkaline-pho
sphatase-cip-in-plasmid-cloning/
describes how CIP has many drawbacks. SAP overcomes some of these
drawbacks and AAP is the best.

It is critical to separate cut DNA from the rest of the gel.
-for getting a good separation of 4-6 kb vectors the author even
recommends using a 0.6% agarose gel
http://bitesizebio.com/articles/cloning-tips-vector-prep/

The author also talks about avoiding exposure of DNA to UV.
"My preferred method is to use a dye such as crystal violet or methylene
blue to visualise the preparative plasmid prep. These stains have a much
weaker interaction with DNA so only bands containing greater than 0.5
micrograms of DNA can be vizualized."
http://bitesizebio.com/articles/cloning-tips-vector-prep/

Some people claim that dephosphorylation is unnecessary if one is doing
directional cloning and two different restriction enzymes are used
Others disagree
Even if you use two different enzymes for digestion, additional
dephosphorylation will reduce the background coming from religation of
the empty vector.
http://www.methods.info/Methods/RNA_DNA/ligation_simple.html

===========================================================================

Here's some info from a troubleshooting document.
www.flemingtonlab.com/Protocols/PlasSubclTroubleGuide.pdf
High numbers of colonies on both the Test and the Control plates (i. e.
usually 100 or
more on the control plate unless cells are very competent).
A) For Non-Directional Strategies.
9) If the strategy is a non-directional approach using phosphatase
treatment of the vector, this high
background is most likely due to inefficient phosphatase treatment.
Solution. Carry out a titration to determine the least amount of
phosphatase treatment needed to
yield vector that gives less than 20 colonies (without insert). Use this
vector sample to ligate to insert.
B) For Directional Strategies. Directional strategies (i.e. the vector is
cut with two different enzymes) should
result in low background since the two incompatible ends cannot religate.
If a high background is observed,
one of the following may be the cause.
10) One of the enzymes used to cut the vector is not digesting or is
inactive.
Solution. Test whether this is a problem by cutting the vector with each
enzyme alone. If one
enzyme is not cutting, the enzyme may have gone bad (typically not
likely), the site may be inhibited by
overlapping dam methylation, the enzyme has significant site specificity,
or the digestion conditions are not
favorable for that particular enzyme. Refer to vendor catalog to
determine whether any of these might be
applicable for the enzyme being used.
11) Although not that likely, it is possible that the DNA sample or some
other solution is contaminated with
uncut plasmid.
Solution. This can be tested by setting up ligation without ligase and
determining the number of
colonies that arise. If this results in a high number of colonies, leave
out solutions one by one until the
contamination is identified and replace appropriate solution.
12) The two restriction sites being digested in the vector are extremely
close together (i.e. 0-3 bp apart).
Many restriction enzymes do not cut well if the site is close to a DNA
end. Therefore, after one enzyme cuts,
the other may not because it is now close to the end.
Solution. In this case, it is best to select different sites in the
polylinker for cloning. If this is not
possible, you may try digesting the vector with one enzyme first and then
the second enzyme (and vice
versa) and set up ligations without insert. One of the enzymes may not be
as susceptible to the end
proximity problem and therefore cutting with the other enzyme first may
allow a more complete digest.
13) There is a mistake in the strategy or the vector map has errors. This
is similar to the issue discussed in
no. 9 above except that in this case, there is a mistake in the vector
plasmid. For example, the vector is cut
with BamHI and HindIII but there is an unaccounted for second HindIII
site or BamHI site on the other side of
the BamHI or HindIII sites, respectively. In this case, although the
vector is digested with BamHI and HindIII,
the vector would be left with either BamHI/BamHI ends or with
HindIII/HindIII ends which would ligate
together without an insert and would result in very high backgrounds.

===========================================================================

azim58wiki: