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Summary Comprehensive exam review EVERYTHING you need to know from student with 91 in course

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Course
Bio 2290

Institution
University Of Western Ontario (UWO )

Comprehensive final exam review: EVERYTHING you need to know from student with 91 in course

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BIO2290 METHODS FINAL
EXAM REVIEW
Monday December 12, 2022

s

,Molecular Biology and Instrumentation
Steps and Biology of Bacterial Cloning
Bacterial cloning: the process by which we have bacteria make numerous identical copies of a
fragment / insert of DNA (contained in a plasmid)
1) Restriction digest: plasmid and insert are digested with the same restriction enzyme(s) to
create compatible ends
2) Ligation: ligase used to “stick” digested insert and plasmid together
3) Transformation: competent E. coli transformed with recombinant plasmid (plasmid
containing insert)
4) Liquid culture growth: colonies containing insert (white) selected and grown in liquid
LB
5) Miniprep: isolate plasmids from bacteria
Downstream applications of cloning
 Use bacteria to produce proteins of interest (ie., insulin)

Calculations/Formulas
Transformation Efficiency (TE)
¿ colonies ( w h ite+blue ) insert plate
TE=
total colonies TE diluted LB plate

 Numerator: total number of bacteria transformed with the plasmid
 Denominator: total number of bacteria that would grow w/o plasmid
o Multiply by inverse of dilution (10-6) to take into account dilution factor
 TE is unitless, and is expressed as a ratio, not a percentage

Ligation Efficiency (LE)
¿ w h ite colonies ( contain insert ) on insert plate
¿=
all colonies ( w h ite+ blue ) insert plate

 Numerator: number of plasmids into which insert was successfully ligated
 Denominator: total number of plasmids (w and w/o insert) transformed into bacteria
 *only the insert plates underwent ligation with insert
 LE is unitless, and is expressed as a ratio, not a percentage

Insert:Vector Ratio
Insert: Vector (plasmid) Ratio
 Molar ratio for the amount of insert relative to the amount of vector (plasmid)
o Ratio calculation influenced by the size (#base pairs) of the plasmid and the
vector due to molar mass
1

, o Often want a 3:1 (insert:vector/plasmid) ratio

Calculate the mass of insert needed, given the mass of the plasmid:

mass insert ( ng )=
(ng plasmid)(¿ kb )
(¿ kb)
×ratio
3
1 ()
Calculate the volume of insert needed, given the concentration and the mass (calculated above):
mass insert (ng)
volume insert ( μL )=
ng
concentratiton insert ( )
μL
%T (Transmission)
P¿
Precent transmittance: %T = × 100 %
Pout

 Pin = input light intensity/power
 Pout = output light intensity/power
Absorbance

A=log ( T1 ), where T (transmittance) is in decimal form
 Unitless
 Measure at λmax
 Linearly linked to concentration
Cell Viability
Live cell count (cells/mL):
 Average 10 live (clear) cell counts for each of the 10 quadrants counted
 Multiply by 250 to go from volume of chamber to mL
 Multiply by 2 to account for dilution with trypan blue dye
 Write cells/mL in scientific notation
Dead cell count (cells/mL):
 Average 10 dead (blue) cell counts for each of the 10 quadrants counted
 Multiply by 250 to go from volume of chamber to mL
 Multiply by 2 to account for dilution with trypan blue dye
 Write cells/mL in scientific notation

% Cell viability=1− ( numbertotal
of dead ( blue ) cells
cells : ¿
live cells+ ¿ dead cells¿ ) ×100 %

2

, Experimental Design and Controls
How to determine/plan the controls you need when designing an experiment:
 Ensure if there is an observed difference it is due to the independent variable
 Control for unexpected effects of any protocols you’re using
 Allows you to determine how many reactions you need to set up (ie., how many tubes)
Start with 2 samples of interest, or sample of interest + experimental control (if there is a
“normal” condition)
Steps:
1. Restriction digest
2. Ligation
3. Transformation
4. Plating
Experimental reaction:
 RD with plasmid
 Ligation with insert
 Transformation of recombinant plasmid into bacteria
 Plate on LB + Amp (selectable marker)
For each type of control, need this same type of control for each sample of interest (unless no
sample of interest present due to that step being controlled for):
1. Ligation technical control (ligation with water, no insert)
2. Transformation technical controls
 RD with TE buffer; Ligation with water
a) Dilute and plate on LB only
b) Plate on LB + amp
3. Plasmid only control
 Plasmid directly into transformation (no RD or ligation), plate on LB + amp

3

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