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
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
