Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis 2 Objective · Digest DNA of pGLO plasmid using restriction endonuclease enzymes. · Run an agarose gel to separate the DNA fragments. Background Restriction enzymes cut DNA at specific sites generating a number of different sized fragments. The size of the fragments will depend on the number of sites the plasmid has and the specific enzyme used. The number of fragments can be predicted by viewing the map of the plasmid Gel electrophoresis is a means of separating DNA in an electrical field. DNA is negatively charged and so will move to the anode (+). Larger fragments will move slower through the agarose matrix than the smaller molecules. Agarose is a polysaccharide polymer derived from seaweed: it is a purified from agar by removing the agaropectin component. Fragments are visualized using ethidium bromide, which will glow orange when exposed to UV light. Materials Restriction digest · Restriction enzymes: Nhe1 and EcoR1 (New England Biolabs) – (KEEP ON ICE) · Plasmid prepared in lab 7 · NanoDrop Lite spectrophotometer · Microfuge tubes – Sterile · 37 C degree bath – block heater · Sterile 10ul and 200ul tips · Bleach bottles for cleaning bench · 10X NE Cut Smart Buffer – comes with enzyme · Nitrile gloves · Sterile DI water · Shaved ice · Ice block for enzymes Gel Electrophoresis · Agarose · Sterile miliQ Water · 15 well comb · 50x TAE buffer · DNA ladder – diluted in sample buffer (1 KB) · Gel loading dye · Gel electrophoresis chamber · Power supply · Ethidium bromide · Gel Sys – visualization system _______________________________________________ Procedure Restriction Digest of plasmid DNA · Safety: Wear nitrile gloves – prevent DNAase from your hands affecting the reaction and protect yourself from ethidium bromide · Clean the bench with bleach - prevents exogenous enzymes interfering you’re your digests. · Use the NanoDrop to determine the amount of DNA in your plasmid prep. Use this information to calculate how much sample you need to pipette into the reaction mix. · Label an Eppendorf tube ‘+’ and another ‘-‘ · Make up a reaction mix in both tubes as follows for one of your plasmid samples · add 1ug of DNA from your plasmid prep · 5ul of 10X NE Cut Smart Buffer · Sterile DI water to make the reaction mix to 50ul For the + tube · DNA x ul · 10X NE Cut Smart Buffer 5ul · Nhe1 (add last to + tube) 1ul · EcoR1 (add last to + tube) 1ul · Sterile DI water To make final volume to 50ul · Add the restriction enzymes last to the + tube ONLY · Repeat with the other two plasmid samples For the – tube · DNA x ul · 10X NE Buffer 5ul · Nhe1 None · EcoR1 None · Sterile DI water To make final volume to 50ul · Do not add any enzyme to the ‘-‘ tube · Repeat with the other two plasmid samples · Mix the tubes by flicking – DO NOT VORTEX · Give a 5 second spin in the centrifuge to bring the contents to the bottom · Incu.

1. Biol 390 – Lab 8 Restriction Digest and Gel Electrophoresis
2
Objective
· Digest DNA of pGLO plasmid using restriction endonuclease
enzymes.
· Run an agarose gel to separate the DNA fragments.
Background
Restriction enzymes cut DNA at specific sites generating a
number of different sized fragments. The size of the fragments
will depend on the number of sites the plasmid has and the
specific enzyme used. The number of fragments can be
predicted by viewing the map of the plasmid
Gel electrophoresis is a means of separating DNA in an
electrical field. DNA is negatively charged and so will move to
the anode (+). Larger fragments will move slower through the
agarose matrix than the smaller molecules. Agarose is a
polysaccharide polymer derived from seaweed: it is a purified
from agar by removing the agaropectin component. Fragments
are visualized using ethidium bromide, which will glow orange
when exposed to UV light.
Materials

2. Restriction digest
· Restriction enzymes: Nhe1 and EcoR1 (New England Biolabs)
– (KEEP ON ICE)
· Plasmid prepared in lab 7
· NanoDrop Lite spectrophotometer
· Microfuge tubes – Sterile
· 37 C degree bath – block heater
· Sterile 10ul and 200ul tips
· Bleach bottles for cleaning bench
· 10X NE Cut Smart Buffer – comes with enzyme
· Nitrile gloves
· Sterile DI water
· Shaved ice
· Ice block for enzymes
Gel Electrophoresis
· Agarose
· Sterile miliQ Water
· 15 well comb
· 50x TAE buffer
· DNA ladder – diluted in sample buffer (1 KB)
· Gel loading dye
· Gel electrophoresis chamber
· Power supply
· Ethidium bromide
· Gel Sys – visualization system
_______________________________________________
Procedure
Restriction Digest of plasmid DNA
· Safety: Wear nitrile gloves – prevent DNAase from your hands
affecting the reaction and protect yourself from ethidium
bromide
· Clean the bench with bleach - prevents exogenous enzymes
interfering you’re your digests.
· Use the NanoDrop to determine the amount of DNA in your

3. plasmid prep. Use this information to calculate how much
sample you need to pipette into the reaction mix.
· Label an Eppendorf tube ‘+’ and another ‘-‘
· Make up a reaction mix in both tubes as follows for one of
your plasmid samples
· add 1ug of DNA from your plasmid prep
· 5ul of 10X NE Cut Smart Buffer
· Sterile DI water to make the reaction mix to 50ul
For the + tube
· DNA
x ul
· 10X NE Cut Smart Buffer
5ul
· Nhe1 (add last to + tube)
1ul
· EcoR1 (add last to + tube)
1ul
· Sterile DI water
To make final volume to 50ul
· Add the restriction enzymes last to the + tube ONLY
· Repeat with the other two plasmid samples
For the – tube
· DNA
x ul
· 10X NE Buffer
5ul
· Nhe1
None
· EcoR1
None
· Sterile DI water
To make final volume to 50ul
· Do not add any enzyme to the ‘-‘ tube

4. · Repeat with the other two plasmid samples
· Mix the tubes by flicking – DO NOT VORTEX
· Give a 5 second spin in the centrifuge to bring the contents to
the bottom
· Incubate for 65 mins in heating block.
· Place the tubes on ice to stop the reaction
Gel Electrophoresis
For one gel
· Block the ends of the gel mold using time tape. Insert the
sample comb for 8 or 15 wells.
· Place the tray in the in the plastic lid box to catch any leaks.
· Weigh out the amount of agarose needed to prepare 50 ml of
1% agarose
· Transfer the agarose to a 250ml Erlenmeyer flask.
· Dilute 1 ml of 50X TAE buffer to 50 ml in a graduated
cylinder and add to the agarose.
· Place a piece of paper towel in the neck of the flask so it
partially blocks the neck.
· Microwave on high for 30 seconds for up to 4 times. USE A
SILICONE HANDGRABBER. Swirl flask gently in between
microwaving. STOP when the agarose is clear and no more agar
powder is visible.
· Cool on bench until it is comfortable to handle.
· Add 0.5ul of ethidium bromide (CAUTION – MUTAGEN) to
the agarose and gently swirl.
· Allow gel to cool so it can be placed against the inside of the
wrist. Running it under cold water helps, but do not let it set.
· Gently pour the agarose into the template mold and let it set
for 10 minutes or more
· Gently remove the comb and the bumpers from the gel.
· Mix 250ml of 1xTAE using your 50X stock
· Place the gel still in the holder in the gel box.
· Pour buffer into the box until it covers the gel.
· Load 10ul of diluted ladder very slowly to the center lane, and
the end lanes

5. · In clean microfuge tubes make up the following.
· For your + and - samples run these in duplicate at different
levels
)
10
2
20
4
· Load the gel as follows:
Lane
Sample vol
Sample
1
10ul
Ladder -1KB
2
10+2 ul
Isolate 1 + enzyme
3
10+2 ul
Isolate 1 - enzyme
4
10+2 ul
Isolate 2 + enzyme
5
10+2 ul
Isolate 2 - enzyme
6
10+2 ul
Isolate 3 + enzyme
7
10+2 ul
Isolate 3 - enzyme
8

6. 10ul
Ladder -1KB
9
20+4 ul
Isolate 1 + enzyme
10
20+4 ul
Isolate 1 - enzyme
11
20+4 ul
Isolate 2 + enzyme
12
20+4 ul
Isolate 2 - enzyme
13
20+4 ul
Isolate 3 + enzyme
14
20+4 ul
Isolate 3 - enzyme
15
10ul
Ladder – 1KB
· Very slowly add each of the + and – digests either side of the
ladder
· Orient the gel so the sample is at the cathode end (black) and
the samples will flow in the direction of the arrow on the side
of the box.
· Place the top on the box and connect the electrodes and turn
the power on.
· Set the voltage to 150 volts and hit run. The display will
show 150 volts and you should see gas coming from the
electrodes.
· Run until the indicator dye is close to the bottom.
· Move the gel to the Gel illuminator and view the bands.

7. Questions
· See the assignment in Canvas
Biol 390-Lab 7 Select transformants and isolate plasmids
4
Objective
· Select transformed cells.
· Freeze cells for future labs.
· Isolate plasmids from transformed cells
Background
p-GLO plasmid
pGLO contains several DNA sequences that enable replication
of the plasmid DNA and expression of the fluorescent trait
(phenotype) in bacteria following transformation. The essential
sequences include the following:
GFP— the jellyfish gene that codes for the production of green
fluorescent protein

8. bla — a gene that encodes the enzyme beta-lactamase, which
breaks down the antibiotic ampicillin. Bacteria containing the
bla gene can be selected by placing ampicillin in the growth
medium.
ori — the origin of pGLO plasmid DNA replication
araC — a gene that encodes the regulatory protein that binds to
the pBAD promoter. Only when arabinose binds to the AraC
protein is the production of GFP switched on.
pBAD promoter — a specific DNA sequence upstream from the
GFP gene, which binds araC-arabinose and promotes RNA
polymerase binding and transcription of the GFP gene.
Multiple cloning site — a region containing restriction sites
(NdeI, HindIII, EcoRI, etc.), sequences that permit the insertion
or deletion of the gene of interest
Materials
For day before
· Three tubes of LB broth / group
· Ampicillin solution (10mg/ml)
· Sterile plastic loops
Day of the lab
· Sterile tips
· Bleach spray bottles
· Nitrile gloves
· 20% glycerol sterile
· Sterile tubes for freezing cultures
· Midisci high speed plasmid mini kit
· Add RNase A to PD1 buffer and store at 4 oC (prepare day
before)
· Check for precipitates in PD2 buffer – if present warm in
37oC bath
· Add 100 ml of absolute ethanol to wash buffer
_______________________________________________
Procedure
Day before

9. · Add ampicillin to LB tubes containing 5 ml of LB broth.
· How much ampicillin do you need to add to make the final
concentration 100ug/ml
· Label LB tubes with your name an number for each colony.
· Inoculate three separate colonies into three different tubes of
LB. Pick three colonies from the LB amp/ara using sterile a
sterile loop for each transfer.
· Incubate tubes at 37oC
Day of lab
· Observe plates from your experiment. Where possible count
the number of colonies. Which colonies glow?
Plate
Observations
+pGLO LB/amp
+pGLO LB/amp/ara
-pGLO LB/amp
- pGLO LB
Freeze cells
· Label 3 sterile Eppendorf tubes with your name and your
colony number.
· Aseptically transfer 0.5 ml of your overnight culture to a
sterile Eppendorf.
· Using a new pipette add 500ul of 20% glycerol
· Freeze at -80oC
High-Speed Plasmid Mini Kit Protocol -Midisci
· Add provided RNase A to the PD1 Buffer and store at 4oC.
(the day before)
· If precipitates have formed in the PD2 Buffer, warm the buffer
in a 37oC water bath, followed by gentle shaking to dissolve.

10. · Add absolute ethanol to the Wash Buffer prior to initial use
(see the bottle label for volume).
· Additional requirements: microcentrifuge tubes.
Step1 Harvesting
· Transfer 1.5 ml of cultured bacterial cells to a microcentrifuge
tube. Microcentrifuge for 1 minute and discard the supernatant.
Step 2 Re-suspension
· Add 200 μl of PD1 Buffer (RNase A added) to the tube and
resuspend the cell pellet by vortex or pipetting.
Step 3 Lysis
· Add 200 μl of PD2 Buffer and mix gently by inverting the
tube 10 times. Do not vortex to avoid shearing the genomic
DNA.
Let stand at room temperature for 2 minutes or until the lysate
is homologous.
Step 4 Neutralization
· Add 300 μl of PD3 Buffer and mix immediately by inverting
the tube 10 times. Do not vortex.
· Microcentrifuge for 3 minutes.
Step 5 DNA Binding
· Place a PD Column in a 2 ml Collection Tube.
· Add the supernatant from Step 4 to the PD Column and
microcentrifuge for 30 seconds.
· Discard the flow-through and place the PD Column back in the
2 ml Collection Tube.
Step 6 Wash
· Add 400 μl of W1 Buffer into the PD Column.
· Microcentrifuge for 30 seconds.
· Discard the flow-through and place the PD Column back in the
2 ml Collection Tube.
Add 600 μl of Wash Buffer (ethanol added) into the PD
Column. Microcentrifuge for 30 seconds.
· Discard the flow through and place the PD Column back in the
2 ml Collection Tube.
Microcentrifuge again for 3 minutes to dry the column matrix.
Step 7 DNA Elution

11. · Transfer the dried PD Column to a new microcentrifuge tube.
· Add 50 μl of Elution Buffer or TE into the center of the
column matrix.
· Let stand for 2 minutes or until the Elution Buffer or TE is
absorbed by the matrix
· Microcentrifuge for 2 minutes to elute the DNA.
· Store at -20oC
Data Analysis
· Explain that in the +pGlo experiments why the colonies that
grew on the LB/amp/ara fluoresced, while those that grew on
LB/amp did not
· Why do colonies of the –Pglo not grow on the LB/amp plate
References
· MIDIsci
· http://shop.midsci.com/protocals/IB47100Protocol.pdf
· Biorad
· http://www.bio-rad.com/en-us/applications-technologies/pglo-
plasmid-map-resources
·
Biol 390-Lab 6 Transformation

12. 2
Objective
· Transform E. coli with the using a plasmid containing Green
Florescent Protein (GFP)
Background
Green fluorescent protein from a jellyfish. This protein has
been engineered into a plasmid. The plasmid also contains an
arabinose promoter that will cause the GFP to be expressed and
a gene that codes for a beta-lactamase; an enzyme that breaks
down ampicillin.
Plasmid and its genes
Materials
For each group
· E. coli starter plate – streaked for single colonies and 24 hours
old
· Agar plates – 1 LB, 2 LB/amp, 1 LB/amp/ara
· Transformation solution – 1 ml color coded
· LB nutrient broth – 1 ml color coded
· Inoculating loops – plastic disposable
· Sterile pipette tips
· Foam tube holder
· Sterile microfuge tubes
· Ice bath
· Stop watch
· Marker
Common work station
· pGLO plasmid
· Heating block – 42oC

13. · Nitrile gloves
______________________________________________
Prelab Preparation
Two days before
· Add 250ul of LB broth to the lyophilized E. coli using a
sterile tip and incubate for 8 to 24 hr at 370C
One day before
· Streak plates for single colonies – incubate 24 to 36 hours.
Each group gets one plate
Day of the lab
· Prepare the pGLO plasmid – transfer 250ul of the
transformation solution into the lyophilized pGLO vial. Mix by
inverting in case there is plasmid DNA attached to the cap.
· Label one tube per group “TR”: aseptically transfer 1000ul to
each of the transformation solution.
· Lab one tube per group “LB”: aseptically transfer 1000ul to
each of the LB nutrient medium.
Procedure
1. Use gloves during this experiment
2. Clean benches with alcohol – be careful
3. Select two sterile microfuge tubes and label
a. +pGLO
b. –pGLO
4. Place in a rack
5. Open tubes and using a separate sterile pipette tip transfer
250ul of the transformation solution (TR) to each tube.
6. Place tubes in foam rack in the crushed ice bath.
a. Note: make sure the tubes are in contact with the ice. You
may have to push theminto the foam.
7. Using a sterile disposable loop transfer 2 to 4 E.coli colonies
from the agar plate to each tube.
a. NOTE – it important to uses between 2 and 4, 1 to 1.5 mm
colonies to maximize the transformation efficiency.
8. Spin the loop in the transformation solution until all the
E.coli have been suspended

14. a. Note there should be no floating chunks.
9. Use a new loop to inoculate the other tube in the same way
using other colonies from the plate.
10. Transfer a 10ul aliquot of the pGLO plasmid to the +pGLO
tube using a sterile tip and an automatic pipette.
a. NOTE – do not add the plasmid to the –pGLO tube
11. Incubate the tubes on ice for 10 mins once the plasmid has
been added.
12. While the tubes are sitting in the ice label your four plates
with your name.
a. +pGLO LB/amp
b. +pGLO LB/amp/ara/
c. -pGLO LB/amp
d. –pGLO LB
13. Transfer the tubes into the heating block set at 42oC for
exactly 50 seconds. Note – timing is critical
14. After exactly 50 seconds quickly transfer the tubes to ice for
2 minutes.
15. Place the tube back in the microfuge tube rack and add
250ul of LB broth to one tube using a sterile pipette tip. Add
250 ul of broth to the other tube using a new sterile pipette tip.
16. Incubate the tubes for 10 min at room temperature.
17. Gently flick the closed tubes with your finger to mix.
a. Note its important to make sure the tubes are mixed. Pipette
100ul from each tubes as follows:
b. +pGlO tube to:
i. +pGLO LB/amp
ii. +pGLO LB/amp/ara
c. – pGlO tube to:
i. –pGLO LB/amp
ii. –pGLO LB
18. Using a sterile loop spread the suspension gently around the
entire surface of the plate. Note this requires very little
downward pressure.
19. Turn plates upside down and incubate for 24 hours at 37oC

15. Biol 390-Lab 5 Preparation for Transformation
2
Objective
· Prepare materials for transformation
· Prepare TAE buffer for electrophoresis in a later lab.
Background
Transformation of bacterial with plasmids is a fundamental
technique in genetic engineering. Cells can be made competent
to take up plasmids by treatment with divalent cations and heat.
Transformed cells will contain a plasmid that contains a gene
that confers resistance to the antibiotic, ampicillin. Therefore,
transformed cells can be selected by growth on LB/amp plates.
Growth on LB/ara /amp will cause the product to be expressed
as the plasmid contains an arabinose promotor
Materials
· LB broth
· LB agar
· Petri dishes

16. Needed when plates are poured
· Ampicillin (amp) solution sterile – 10 mg/ml stock
· Arabinose (ara) solution sterile – 200 mg/ml stock
For TAE (Tris –acetic acid-EDTA) buffer
· Tris base
· Glacial Acetic acid
· EDTA
_______________________________________________
Procedure
Make LB agar
You will need per group
· 1 LB plates
· 2 LB/ amp plates
· 1 LB /amp/ara
How much volume each will you need for the whole class?
How much does a petri dish hold?
Make up separate bottles of LB agar for each set of the plates
Formula for Luria broth.
· Luria broth – 20g/l
· Agar – 15g/l
Heat the broth agar mixture until the agar melts (about 95oC).
Your bottles will be autoclaved at 121oC for 20 minutes and
stored until 2 days before.
As arabinose or ampicillin can not be autoclaved, sterile
solutions of these will be added to the agar after it is re-melted
2 days before the experiment.
TAE Buffer
You need to prepare 200ml of a 50X stock of TAE buffer to use
for gel electrophoresis
Recipe for one liter of 50X stock ‘
· Dissolve 242g Tris base in water
· add 57.1mL glacial acetic acid,
· add 18.6g EDTA solution
· bring the final volume up to 1 liter.

17. This stock solution will be diluted 50:1 with water to make a
1X working solution. The 1X solution will contain 0.4mM Tris,
20mM acetic acid, and 1mM EDTA.