A PRACTICAL SET OF RECOMMENDATIONS FOR LIGATION

The recommendations below define a reasonable compromise set of conditions for efficient ligation of vector into C1,1 product, made possible by the demonstrated breadth of the ligation optima (i.e. being off by -+ 30% in insert concentration makes little difference in final yield). We presume here that the most commonly used plasmid cloning vectors range from 2.5 to 7.5 kb, and that inserts to be cloned are generally smaller than 10 kb.

1. Whenever possible, use either forced directional cloning or phosphatased vectors.

2. Under normal conditions, keep the vector concentration at 1 ug/ml or less. If the primary intent is to recover with maximal efficiency "infinitesimal" amounts of insert, then higher concentrations of vector may be optimal.

3. Assuming symmetric ligation with unphosphatased vectors:

a) If the insert is large (i.e. > 800 bp), use 8ug/ml of insert

b) If the insert is small (i.e. < 200 bp), use 1nM insert for a 7.5 kb vector or 5 nM insert for a 2.5 kb vector.

c) If the insert is intermediate sized, optimum conditions are intermediate. As a starting point, try using 5 nM insert for a 7.5 kb vector or 10 nM insert for a 2.5 kb vector, increasing or decreasing this trial concentration according to whether the insert more closely resembles a large or small fragment.

4. Assuming asymmetric ligation (i.e. forced directional cloning):

a) Regardless of insert size, use a 3:1 insert:vector molar ratio.

b) However, when following the above rule, do not exceed 5µg/ml of insert.

5. Assuming symmetric ligation with phosphatased vector:

a) If the insert is large (i.e. > 800 bp), use 8µg/ml of insert.

b) If the insert is small (i.e. < 200 bp), use a 3:1 insert:vector molar ratio

c) If the insert is intermediate sized, optimum conditions are intermediate. As a starting point, try using 5 nM insert for a 7.5 kb vector or 10 nM insert for a 2.5 kb vector, increasing or decreasing this trial concentration according to whether the insert more resembles a large or small fragment.

6. If the intent of the ligation is to recover "infinitesimal" amounts of insert, note that forced directional ligation, if possible, offers the best possible yields, followed by ligation with excess phosphatased vector.

7. The above recommendations hold when the intent of the ligation is to yield circular products. For lambda and cosmid ligations, where the intent is to obtain linear concatamers, insert and vector should be ligated at a 1:1 molar ration at as high a concentration as practical.

PROMEGA SUGGESTIONS

It is recommended that a 1:1, 1:3 or a 3:1 molar ratio of vector:insert be used when cloning into one of the Riboprobe system plasmid vectors (2 to 3.5 kb). The table below illustrates the conversion of molar ratios to mass ratios for a 3.0 kb plasmid and a 0.5 kb DNA fragment which is to be inserted.

Calculation of Vector and Insert DNA Ratios

In this example the mass ratio of the vector to the insert is 3.0:0.5 or 6:1

Vector Insert

1:1 molar ratio 1.0µg 0.167µg

1:3 molar ratio 1.0µg 0.501µg

3:1 molar ratio 1.0µg 0.056µg

Incubation times should be as follows:

Overnight at 40.

4-6 h at 150.

1 h at 250.

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