Tube DNA

A and D vector (30 fmoles [approx. 100 ng]) B insert (foreign) (30 fmoles [approx. 10 ng])

C and E vector (30 fmoles) plus insert (foreign) (30 fmoles) F superhelical vector (3 fmoles [approx. 10 ng])

The molar ratio of plasmid vector to insert DNA fragment should be approx. 1:1 in the ligation reaction. The final DNA concentration should be approx. 10 ng/pl.

10x Ligation buffer 1.0 pl

Bacteriophage T4 DNA ligase 0.1 Weiss unit

10x Ligation buffer 1.0 pl

H2O to 10 pl no DNA ligase

The DNA fragments can be added to the tubes together with the H2O and then warmed to 45°C for 5 minutes to melt any cohesive termini that have reannealed during fragment preparation. Chill the DNA solution to 0°C before the remainder of the ligation reagents are added.

6. Incubate the reaction mixtures overnight at 16°C or for 4 hours at 20°C.

7. Transform competent E. coli with dilutions of each of the ligation reactions as described in Chapter 1, Protocol 23 , Chapter 1, Protocol 24 , Chapter 1, Protocol 25 , or Chapter 1, Protocol 26 . As controls, include known amounts of a standard preparation of superhelical plasmid DNA to check the efficiency of transformation.

Tube

DNA

Ligase

Expected number of transformed colonies

A

vector

+

approx. 0 (approx. 104 fewer than Tube F)

B

insert

+

0

C

vector and insert

+

approx. 10-fold more then Tube A or D

D

vector

-

approx. 0 (approx. 104 fewer than Tube F)

E

vector and insert

-

some, but fewer than Tube C

F

superhelical vector

-

>2 x 105

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