Lab Report I (Biol 203, Spring 2020) NAME (First, LAST):
Sessions 1,2,4,5,6,7
PART I (No lab data required) 60 pts.
1. In the 203 laboratory, you have used an enzyme called T4 DNA ligase. Answer
the few questions bel
...
Lab Report I (Biol 203, Spring 2020) NAME (First, LAST):
Sessions 1,2,4,5,6,7
PART I (No lab data required) 60 pts.
1. In the 203 laboratory, you have used an enzyme called T4 DNA ligase. Answer
the few questions below regarding this enzyme:
a) what is the definition of one Unit for this enzyme? Well, the manual says we are
using 20 units of T4 DNA Ligase. It doesn’t define what a CEL unit is and I had
to look it up online at the manufacturer website. “One unit is defined as the
amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA
(5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction
volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer.”
Source.
b) what are the optimal reaction conditions recommended by the manufacturer
(buffer and incubation conditions)? Did you use these conditions in lab?
Temperature of 16 degrees Celsius and “1X T4 DNA Ligase Reaction Buffer.”
Yes, we used these conditions since 1/10 x 10 = 1x.
c) can the enzyme be inactivated? If so, how? It can be inactivated at high enough
heat (65C) for ten minutes.
d) in the protocol recommended by the manufacturer (NEB):
-what is the recommended reaction volume? 20 microliters.
- how many units of enzyme (at 400,000 U/ml) per reaction are recommended? 400
units.
-what is the molar ratio of vector: insert? Which is in excess? 1:3 with insert in
excess.
-do blunt end versus sticky end ligations required the same incubation time? Why or
why not? No they don’t. Cohesive ends require 10 minutes at room
temperature while blunt ends require 120 minutes at room temperature. This
is because blunt end ligation is less efficient.
- here is a model showing the reaction catalyzed by T4 DNA ligase (from Lewin,
Genes VIII, Pearson publ.)
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