2. Laboratory 2: How do you begin to clone a gene?
LSSI Alum,
Lindsey Engle
LSSI Alum,
Shawn Hurley
LSSI Alum,
Mary Haus

3. Pre-Assignment http://www.dnaftb.org/34/animation.html
Great background animated storyboard
Could assign as HW if one-to-one
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
**Please be aware, sticky ends are not identified correctly, but overall process is.

4. Resource Links/Additional Background

5. Important Vocabulary/Terms
-Plasmid
-Vector
-Insert
-Construct
-Digest
-Ligase
-Palindromic
-Restriction Enzyme
-Sticky Ends
-Complementary Bases
-Origin of replication
-Promoter
-Antibiotic resistance gene
-Regulatory gene

6. Complete Genetic Engineering Sequence
Lab 1: Tools of the Trade – Pipetting and Gel Electrophoresis
Lab 2: Restriction Digest (cut pKAN-R and pARA)
Lab 3: Ligation (combine pieces to make pARA-R)
Lab 4: Confirmation (gel electrophoresis)
Lab 5: Transformation (introduce pARA-R into bacterial host cells)
Lab 6: Culture transformed bacteria, isolate and purify the protein

7. Goals of Lab 2
Logistical (students will coordinate procedural steps necessary to):
Perform restriction digest to generate DNA fragments for future cloning and expression of the red fluorescent protein gene in bacteria
Educational (students will be able to):
Identify the common characteristics of plasmids
Explain how plasmids are used as vectors in gene cloning/expression
Describe the function of restriction enzymes
Explain restriction enzymes are used to create recombinant plasmids

8. What is a plasmid?
Comparatively small circular double-stranded DNA molecules of bacterial origin
Range in size from 1,000 to 200,000 base pairs (bp)
Independent of bacterial chromosome, carrying “nonessential” genes

9. Antibiotic resistance Multiple cloning site (polylinker)
Function:
- Region responsible for initiating the copying of plasmid DNA
- Site to which RNA polymerase binds to begin transcription
- Gene coding for a product that confers antibiotic resistance
- Unique restriction sites allow for the digestion of plasmid & introduction of insert (foreign gene)
- Gene coding for a product that regulates transcription of the insert
Plasmid Features:
Origin of replication
Promoter
Antibiotic resistance
Multiple cloning site (polylinker)
Regulatory element(s)

10. promoter
regulatory
gene
MCS
(with insert)
antibiotic
resistance
gene
origin of replication (Ori)

11. Plasmids as Vectors

12. What are restriction enzymes?
Catalytic proteins that function like molecular scissors, cutting double-stranded DNA at distinct recognition sites that are usually unique to a particular enzyme.

13. What are restriction enzymes?
Characteristics:
Recognition sites are palindromic sequences, usually 4-8 nucleotides in length
5’ – G A A T T C – 3’
3’ – C T T A A G – 5’
Cleave covalent bonds of sugar-phosphate backbone
If enzyme is a staggered cutter, generates sticky ends (unpaired overhangs capable of hydrogen bonding with complementary bases) 5’ – G A A T T C – 3’
3’ – C T T A A G – 5’
Nonemclature based on source bacterial species & strain E co R I
1st letter
of genus
(Escherichia)
roman numeral designates
order of discovery
1st two
letters of species (coli)
strain

14. Application of These Molecular Tools
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 cutting each DNA sample with same restriction enzyme(s))

15. 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 with promoter sequence (pBAD) and an antibiotic resistance gene for kanamyacin (kanR)
pARA – has an antibiotic resistance gene for ampicillin (ampR) and a regulatory gene (araC), coding for arabinose activator protein
Arabinose is a sugar that induces transcription of the rfp gene by working with arabinose activator protein
Will use 2 restriction enzymes on each plasmid allowing the segment from pKAN-R to later be inserted into the pARA plasmid
BamHI
HindIII

16. Lab 2 – Creating the Digest

17. Restriction Digest Fragments
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

18. 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

19. Lab & Aliquoting Guide-Lab 2
Reagents/Supplies
Aliquot
Storage Temp
Notes
1.4 ml 2.5X Buffer/class (2.5xB)
20ul/group 4o
110 ul of pKAN/class (pKAN)
10ul/group
-20o
110 ul of pARA/ class (pARA)
65 ul of Restriction Enzyme/class (RE)
5ul/group
12mL of DI water/ kit (dH20)
1ml/group RT
Keep this for all Labs
Equipment/Supplies
10 Student boxes with the following:
1 p20 micropipette microfuge rack
1 p200 micropipette bag of microfuge tubes
1 p1000 micropipette bag of microfuge tubes
1 waste and box of refillable tips (2 ul-200 ul)
1 ice bucket
4 Mini centrifuges
1 Water bath
2 Floating racks

20. 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

21. 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
Put all tubes in floating rack and set in 37oC water bath for 60 mins.*
Centrifuge all tubes
* - if time is limited, reducing the digest time to minutes is aaceptable
Remove and place tubes into freezer overnight

22. Teacher Video Resources
Mixing two solutions video:
Digestion video (different digest, but good techniques):
How restriction enzymes work (good, short):
Longer, overall of lab 2 created for absent students (screen cast):
Fun one from MIT. Covers whole process 1st half good for Labs 2&3:
***remember, sticky end issue here