Worksheet 4_ Attempt review La Trobe University BIOMEDICAL MED3LAB
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Module
MED 3LAB BIOCHEM & MEDICAL BIOLOGY
Institution
MED 3LAB BIOCHEM & MEDICAL BIOLOGY
Worksheet 4: Attempt review
My LMS Subjects / 2021-PSB-MED3LAB(SP-T3-PT) / Assessment / Worksheet 4
Started on Thursday, 28 October 2021, 12:05 AM
State Finished
Completed on Friday, 29 October 2021, 5:41 PM
Time taken 1 day 17 hours
Grade 19.50 out of 21.50 (91%)
Information
Laboratory Mo...
worksheet 4 attempt review httpslmslatrobeeduaumodquizreviewphpattempt12167052ampcmid5107028 121 my lms subjects 2021 psb med3labsp t3 pt assessment worksheet 4 started on thu
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11/29/21, 2:01 AM Worksheet 4: Attempt review
My LMS Subjects / 2021-PSB-MED3LAB(SP-T3-PT) / Assessment / Worksheet 4
Started on
Thursday, 28 October 2021, 12:05 AM
State
Finished
Completed on
Friday, 29 October 2021, 5:41 PM
Time taken
1 day 17 hours
Grade
19.50 out of 21.50 (91%)
Information
Laboratory Module 4 Assessment
In this laboratory module, you prepared plasmid DNA (i.e. performed a “miniprep”) from overnight cultures of positive
GFP-producing and negative non-GFP-producing transformed cells before subjecting it to restriction digestion with
BamHI and HindIII. The results of the digests were compared after separating the DNA fragments on agarose gel
electrophoresis and observing under UV illumination in the presence of SYBR Safe dye. This is a typical experimental
approach to determining transformation status without relying on the colour and fluorescence of transformed colonies
on the plate (as is possible for GFP).
You also set up an overnight culture of the pQE30-GFP transformed cells and used it for induction and expression of
hexahistidine-tagged GFP. A time course of the induction process was carried out by collecting cell samples prior to and
1 and 2 h after the addition of the inducer. The bulk of the induced cells was then harvested and the hexahistidine-
tagged GFP protein was purified using immobilised metal affinity chromatography (IMAC) and analysed by sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
The following quiz will probe your understanding of the theoretical and practical aspects of this practical module.
The figure above outlines the major steps involved in the purification of plasmid DNA using the alkaline lysis
method and the reagents and spin columns from the Wizard® Plus SV Minipreps DNA Purification System.
Identify why each of the chemicals is included from the available options.
EDTA:
To chelate the Mg2+ ions that are required for the nuclease activity of DNases
RNase A:
To degrade the cellular RNA present in the cells following lysis
NaOH:
To disrupt hydrogen bonding, thus temporarily denaturing dsDNA - an initial step to allow plasmid DNA purification
SDS:
To solublise cell membranes and denatures bacterial proteins, including damaging DNases
Alkaline protease:
Inactivates endonucleases and other proteins released during cell lysis that can adversely affect the quality of the
Potassium acetate:
To decreases the pH of the sample, thus enabling hydrogen bonds between two DNA strands of the plasmid to refor
The figure above outlines the major steps involved in the purification of plasmid DNA using the alkaline lysis
method and the reagents and spin columns from the Wizard® Plus SV Minipreps DNA Purification System.
Identify why each of the chemicals is included from the available options.
EDTA: [To chelate the Mg2+ ions that are required for the nuclease activity of DNases]
RNase A: [To degrade the cellular RNA present in the cells following lysis]
NaOH: [To disrupt hydrogen bonding, thus temporarily denaturing dsDNA ‑ an initial step to allow
plasmid DNA purification]
SDS: [To solublise cell membranes and denatures bacterial proteins, including damaging DNases]
Alkaline protease: [Inactivates endonucleases and other proteins released during cell lysis that can adversely
affect the quality of the isolated DNA]
Potassium acetate: [To decreases the pH of the sample, thus enabling hydrogen bonds between two DNA strands
of the plasmid to reform (i.e. renature) ]
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