Ligation

Question 1

Ligation Reaction [why is ligation necessary. what enzyme is used, what happens chemically? What is the required co-factor and why? How much ATP is consumed for each phosphodiester bond made]

Answer 1

-foreign DNA inserted into vector through ligation
-DNA ligase is used (for lab we use t4 DNA ligase)
-Joining linear DNA fragments together with covalent bonds.
More specifically, DNA ligation involves creating a phosphodiester
bond between the 3’ hydroxyl group of one nucleotide and the 5’
phosphate group of another.
-ATP is a cofactor because it adds a necessary adenylyl leaving group so that ligation can occur
-1 ATP consumed per each phosphodiester bond. Insertion of foreign DNA needs 2 equivalents of ATP.

Question 2

Ingredients for Ligation (4 ingredients and optimal temperature for T4 Ligase)

Answer 2

-water
-compatible DNA fragments
-buffer containing ATP
-T4 DNA Ligase (~0.01 units for sticky ends, ~1 unit for blunt end)
- 16ºC

Question 3

Reagents for Ligation Experiment

Answer 3

-Digested HindIII by same restriction enzyme (DNA to be lighted) [4 uL]
-10x ligation buffer (to keep pH range for enzymes also has ATP) [2.5 uL]
-T4 ligase (enzyme) [3 uL]
-deionized water (to bring up volume) [15.5 uL]