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-phosphatase-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.