LABORATORY 2 & 3: HOW DO
YOU BEGIN TO CLONE A
GENE?
LSSI Alum,
Mary Haus
Pre-Assignment
http://www.dnaftb.org/34/animation.html
• Great background
animated storyboard
– Could assign as HW if one-toone
– Could also complete as pre-lab
in computer lab or if you have
class sets of tablets or
chromebooks
– Could complete together in
class using teacher computer
https://www.youtube.com/watch?
v=nfC689ElUVk
**Please be aware, sticky ends are
not identified correctly, but overall
process is.
What is a plasmid?
• Definition: Small,
circular DNA molecules,
ranging in size from
1,000 to 200,000 base
pairs.
• Characteristics:
– Can make copies of itself
independently of bacterial
chromosome using the ori
site for initiation.
– Can initiate transcription
using its promoter
sequence.
– Often carry antibiotic
resistance genes.
– Can be passed through
bacterial conjugation.
Plasmid Features:
• Ori (replicating/cloning)
• Promoter
• Antibiotic resistance
• Restriction sites
What are restriction enzymes?
• Definition: Proteins
that restrict the growth
of bacteriophage by
recognizing and
destroying the phage
DNA without damaging
the host (bacterial)
DNA.
• Characteristics:
Different strains of
bacteria possess
restriction enzymes that
cut at specific DNA
sequences called
recognition sites and
often create sticky ends.
What are restriction enzymes?
• Importance of restriction enzymes and sticky ends:
– Scientists can build designer plasmids that contain specific
restriction sites
– This allows scientist to cut out and recombine genes to allow for
cloning and gene expression. (requires sticky ends)
– Sticky ends: want to form hydrogen bonds which scientists use
to ligate the DNA together again in desired combinations.
Lab 2 – Creating the Digest
(cutting up the DNA)
• Purpose: to produce the DNA fragments that will be
joined to make the recombinant plasmid.
– Will need to cut two plasmids
• pKAN-R – has the rfp gene, an antibiotic resistance gene for
kanamyacin (kan-R), and the promoter sequence (pBAD)
• pARA – has an antibiotic resistance gene for ampicillin (ampR)
and the arabinose activator (araC)
– Arabinose is a sugar that is needed by the promoter to begin
transcription of the rfp
– Will use 2 restriction enzymes on each plasmid allowing
the segment from pKAN-R to later be inserted into the
pARA plasmid
• BamHI
• HindIII
Lab 2 – Creating the Digest
Lab 2 – Creating the Digest
Restriction Digest Fragments
BamH I
Hind III
4,706 bp
BamH I
Hind III
4,496 bp
Hind III
BamH I
806 bp
Hind III
BamH I
376 bp
Safety-Lab 2
• Use laboratory coats, safety glasses and gloves as
appropriate
• Avoid restrictive clothing and open-toed shoes
• No eating or drinking in the lab
• Make sure that students are familiar with the
operating instructions and safety precautions
before they use any of the lab equipment
• Check all MSDS (Material Safety Data Sheets) for all
chemicals and reagents in the lab before preparing
and running the lab
www.amgenbiotechexperience.com
Lab & Aliquoting Guide-Lab 2
Reagents/Supplies
1.4 ml 2.5X Buffer/class (2.5xB)
Aliquot
20ul/group
Storage Temp
4o
110 ul of pKAN/class (pKAN)
10ul/group
-20o
110 ul of pARA/ class (pARA)
10ul/group
-20o
65 ul of Restriction Enzyme/class
(RE)
5ul/group
-20o
12mL of DI water/ kit (dH20)
1ml/group
RT
Equipment/Supplies
10 Student boxes with the following:
1 p20 micropipette
1 p200 micropipette
1 p1000 micropipette
1 waste and
1 ice bucket
4 Mini centrifuges
1 Water bath
2 Floating racks
www.amgenbiotechexperience.com
1 microfuge rack
1 bag of microfuge tubes
1 bag of microfuge tubes
1 box of refillable tips (2 ul-200 ul)
Notes
Keep this for all Labs
Completing Lab 2
Teacher Tips –
*make sure students label with
initials/group
*Remind them of mixing techniques
*Make sure centrifuge is balanced
before running
*Identify for students when to use a
new tip
Completing Lab 2
(pg. B-15 in teacher manual)
Label tubes
Add 4.0 uL 2.5xB into each
Add 4.0 uL K into K+ and
K- tubes
Add 4.0 uL A into A+ and
A- tubes
Add 2.0 uL RE into K+
and A+ tubes and mix
Add 2.0 uL dH2O in K- and
A- tubes and mix
Centrifuge all tubes
Put all tubes in floating
rack and set in 37oC water
bath for 60 mins.
Remove and place tubes
into freezer overnight
Teacher Video Resources
• Mixing two solutions
video:
https://www.amgenbiotechexperience.
com/curriculum/curriculumresources/mixing-two-solutions
• Digestion video (different
digest, but good
techniques):
https://www.youtube.com/watch?v=Gs
Wo8dCivWs
• How restriction enzymes
work (good, short):
https://www.youtube.com/watch?v=lW
XryzgRces
• Longer, overall of lab 2
created for absent
students (screen cast):
https://www.youtube.com/watch?v=4
wbStjWEM8A
• Fun one from MIT. Covers
whole process 1st half
good for Labs 2&3:
•
https://www.youtube.com/watch?v=nf
C689ElUVk
***remember, sticky end issue here
What are Ligases?
• Definition: Enzymes that • Role in Gene Cloning:
catalyze the formation of
When the unpaired bases
covalent bonds in the
of two sticky ends come
sugar phosphate
together and form
backbone.
Hydrogen bonds, the DNA
Ligase catalyzes the
• Ligation is basically the
formation of the covalent
reaction that joins two
bonds between adjacent
pieces of DNA together.
nucleotides.
Lab 3 – Building the Recombinant
Plasmid
• Purpose: to ligate the DNA fragments you produced
during Lab 2 to make new recombinant plasmids.
– There are four fragments that will be recombining
– There are 10 different possible plasmids produced, each
with different combinations (have students predict all
possible combinations – key is in teacher guide)
– The plasmid of interest is the one containing rfp, pBAD,
ampR, and araC
• The recombinant plasmid is called pARA - R
Recombinant plasmid of interest
Safety-Lab 3
• Use laboratory coats, safety glasses and gloves as
appropriate
• Avoid restrictive clothing and open-toed shoes
• No eating or drinking in the lab
• Make sure that students are familiar with the
operating instructions and safety precautions
before they use any of the lab equipment
• Check all MSDS (Material Safety Data Sheets) for all
chemicals and reagents in the lab before preparing
and running the lab
www.amgenbiotechexperience.com
Lab & Aliquoting Guide-Lab 3
Reagents/Supplies
Aliquot
Storage Temp
30 ul Ligase/class (Lig)
2ul/group
-20o
50 ul 5xB (Ligase B)/ class (5xB) 5ul/group
-20o
Equipment/Supplies
10 Student boxes with the following:
1 p20 micropipette
1 p200 micropipette
1 p1000 micropipette
1 waste and
1 ice bucket
4 Mini centrifuges
1 Water bath
2 Floating racks
www.amgenbiotechexperience.com
1 microfuge rack
1 bag of microfuge tubes
1 bag of microfuge tubes
1 box of refillable tips (2 ul-200 ul)
Notes
Completing Lab 3
Teacher Tips –
*make sure students label with
initials/group
*Remind them of mixing techniques
*Make sure centrifuge is balanced
before running
*Identify for students when to use a
new tip
Completing Lab 3
(pg. B-33 in teacher manual)
Check reagents
Place K+ and A+ into 70oC
water bath for 30 mins
Label LIG tube with group
and class period
DIY Water
Bath
• ***complete paper
plasmid activity (see
next slide)
Paper Plasmid Activity
(starts on Pg. 43 in student manual)
Modeling Activity: Choose
the appropriate restriction
enzyme to use to insert the
insulin gene
Completing Lab 3
(continued from previous slide)
Remove K+ and A+ from
water bath
Add 4.0 uL K+ into LIG
Add 4.0 uL A+ into LIG
Add 3.0 uL 5xB into LIG
Add 2.0 uL dH20 into LIG
Mix reagents in LIG
Centrifuge LIG tube
Place LIG, K+, and A+ in
rack
Incubate at room
temperature overnight
Teacher Resources
• Making a recombinant
(good, short clip):
https://www.dnalc.org/view/15476Mechanism-of-Recombination-3Danimation-with-with-basicnarration.html
• Recombinant DNA
animation:
http://www.bioteach.ubc.ca/Teachin
gResources/Applications/GMOpkgJKl
oseGLampard2.swf
• Overall process – Good
short animation:
http://www.pbslearningmedia.org/re
source/biot11.sci.life.gen.genengdna
/genetic-engineering-and-workingwith-dna/
• Recombinant DNA
quizlet:
https://quizlet.com/7435136/recomb
inant-dna-technology-flash-cards/
Note on Sequencing
Suggested sequences are included in your teacher guides,
lessons today were not necessarily presented in this order.
• Lab 2 Sequence (pg. B4)
• Session 1 – Review,
questions, and
discussion
• Session 2 – “Clone that
Gene” paper plasmid
activity and questions
• Session 3 – Complete
lab 3
• Lab 3 Sequence (p B-24)
• Session 1 – Review and
questions
• Session 2 – Complete
lab 3