Cohesive?End?Ligation

1) The ligation mixture contains the following:

vector (~100 ng)
　　insert (equimolar or 2 or 3 X molar concentration of vector)
　　water added to 18 µl

2) Heat the mixture at 45 ℃ for 5 min. to melt any annealed cohesive ends.

3) Cool on ice.
　　Add ligase buffer (+ATP) 2 µl
　　ligase 0.5 µl

4) Incubate reaction mixture at 14 ℃ for 2 - 4 hours or overnight at 4 ℃.

5) Heat ligation mixture at 65 ℃ for 10 minutes to deactivate the ligase.

6) Use 5 - 10 µl of the ligation mixture to transform competent cells.

Note:

a) In order for the to circularise, it is important not to use too high a concentration of (high concentration of promotes intermolecular reaction and therefore forms long linear molecules). Concentration of vector should normally be <10 ng/µl. Try diluting the 5X and then further dilute this 5X to give 3 reaction mixtures at different concentration. Calculation to obtain the best vector and insert concentration is normally not required.

b) Do a control ligation of the vector without any insert. If the background is low, there is no need for screening of the transformed cells for vector with ligated insert.

c) For blunt end ligation (which is much less efficient compare to cohesive end ligation), use a higher concentration of and ligase (~10 X). Higher concentration of ligase (~5x) is also useful for sticky end ligation if ligation time is short.

d) Only use phosphatase when strictly necessary. Fewer steps means less problem. If it is necessary to use it, choose SAP (shrimp alkaline phosphatase - easier to deactivate) instead of CIP. Only use the recommended amount as too much can damage the ends of (this is very important - damaged ends won't ligate).

e) Avoid using TBE when gel-purifying (use TAE instead). Also use the best grade agarose or low m.p. agarose for the gel. Contaminants (such as sulphated agarose) can affect your ligation efficiency. should always be gel-purified before ligation.

f) Some enzymes have been suggested to be able to stick to the ends of thereby affecting ligation. There is uncertainty if this is the case, or if it is a problem of enzyme carrying over during purification. In such cases the solution needs to be treated with proteinase K followed by phenol/chloroform . Taq polymerase, for example, may not be completely removed during purification and can fill in the digested end of and thereby preventing ligation. Restriction enzymes can also digest ligated . Gel purification, however, should remove the restriction enzymes well.

g) Keep the buffer in small aliquots as the freezing/defrosting cycles may damage the ATP. Also do not use too high a concentration of ATP (max. 0.5mM) for blunt-end ligation as polyadenation of blunt-ended may occur?

h) The ligase works best at higher temperature but the reaction temperature is kept low to allow the ends of the to anneal together. The Tm of the ends are generally ~ 12-16 ℃ , but EcoRI may be lower (5-6 ℃ ) since ends are AATT. (For blunt-end ligation, the temperature may not be so important, ligation at higher temperature may therefore be more desirable.

i) Avoid use condensing agents like hexaminecobalt chloride especially for sticky end ligation. They promote intermolecular reaction therefore form concatamers rather than closed plasmids. However, if you must use them, make sure there's K⁺ in the buffer which promote circularisation of plasmid.

j) One useful idea in blunt-end ligation, add restriction enzymes in the ligation mixture - normally blunt-end ligation of insert to vector does not reform the restriction site (therefore when you design the oligo that make sure it doesn't reform the restriction site when ligated), therefore only religated vector is digested. See one-tube PCR cloning for an example.

k) Do remember to polish off the ends after PCR if you're doing blunt-end ligation of PCR product produced by Taq polymerase. However, there's no adenylation for other polymerases like Tli.

l) Use only highly competent cells for transformation in cloning work. Expect 50-1000 transformants (if things go well that is).

m) It is possible to clone gene without doing ligation. Basically, you add extra 11-12 bases LIC site (ligation independent cloning site).The PCR products are digested in the presence of T4 polymerase with dTTP or dATP which expose the LIC site when can annealed with another LIC site on the vector (available from Pharmingen and Novagen) which can then be transformed.See Haun et al, Biotechniques 13(4)515-8,1992,Rashtchian,Current Opinion in Biotechnology 6:30-36,1995

n) Make-your-own TA-cloning vector - can use plasmid pDK101 [ATCC 77406], a derivative of plasmid pGEM[R]5fZ(+). Digest with the endonuclease XcmI which give the overhangs for cloning. Another way is to digest pBluescript with EcoRV and perform a Taq polymerase 3'-terminal addition in the presence of dTTP.See TIBS article "Cloning PCR products using TA vectors " for more discussion on TA-cloning and also other protocols sites to making T vector.