Worksheet 2: Attempt review
My LMS Subjects / 2021-PSB-MED3LAB(SP-T2-FT) / Assessment / Worksheet 2
Started on Saturday, 12 June 2021, 12:55 AM
State Finished
Completed on Tuesday, 15 June 2021, 10:59 AM
Time taken 3 days 10 hours
Grade 24.75 out of 25.50 (97%)
Information
Worksheet - Cons...
My LMS Subjects / 2021-PSB-MED3LAB(SP-T2-FT) / Assessment / Worksheet 2
Started on
Saturday, 12 June 2021, 12:55 AM
State
Finished
Completed on
Tuesday, 15 June 2021, 10:59 AM
Time taken
3 days 10 hours
Grade
24.75 out of 25.50 (97%)
Information
Worksheet - Constructing the pQE30-GFP expression vector
In this exercise, you relate both the theoretical and practical aspects of the protocols used for restriction
enzyme digestion, DNA purification, gel electrophoretic analysis and ligation of the pQE30 and GFP cDNA.
Please read and follow any instructions carefully as these questions will be automatically graded.
PART A: Restriction Enzyme Digestion & Purification of Digested DNA
The restriction endonucleases used in this practical are purchased from a commercial company. These are supplied
at 10 U/μl in 50% (v/v) glycerol.
Based on the protocols used in Laboratory Module 2:
The digestion reaction contained a total of
40
U of restriction enzyme
The final concentration of glycerol in the digestion reaction was
8
%
TIP: Be sure to understand what 1 U (unit) of restriction enzyme means in answering this question.
Note. Your answers should be to one decimal place only. Do not include units as a part of your answer.
There was a total of 4 μl of restriction endonucleases used in the 5 μl of BamHI/HindIII premix.
Each 5 μl aliquot contained 2 μl of each restriction enzyme plus 0.5 μl of 10 x Buffer E and 0.5 μl of water.
As each μl of RE provides 10 U of enzyme, there is 4 μl x 10 U = 40 U of restriction enzyme in the digestion reaction.
---
The restriction endonucleases are stored in 50% (v/v) glycerol (C1)
Volume of RE used (V1) = 4 μl
Final reaction volume (V2) = 25 μl
Therefore, the final concentration of glycerol (C2) = 4 ul/25 ul x 50% = 8%
Given the total units of BamHI included in the pQE30 digestion reaction and the specified incubation time, what is
the theoretical amount of DNA that could be digested under these conditions?
Select one:
1 μg
2 μg
4 μg
10 μg
20 μg
25 μg
40 μg
50 μg
Your answer is correct.
As 2 μl of BamHI (20 units) was used for the digestion of the pQE30 vector, which was performed for 2 h, the theoretical
amount of DNA that could be cut was = 20 μg/h x 2 h = 40 μg of DNA can be theoretically cut.
Definition: One unit (U) of restriction endonuclease activity is defined as the amount of enzyme required to produce a
complete digest of 1 µg of substrate DNA in 1 h (in a total reaction volume of 50 µl) under optimal assay conditions for the
restriction endonuclease.
Note that while we may have used a 25 µl reaction volume, this may not be optimal but does not halve the restriction
endonuclease activity.
The correct answer is:
40 μg
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