2. Laboratory 2A: How do you begin to clone a gene?
LSSI Alum,
Shawn Hurley

3. Resource Links/Additional Background

4. 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

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

6. Goals of Lab 2A
Logistical (students will coordinate procedural steps necessary to):
Perform restriction digest on recombinant plasmid (pARA-R) in order to indirectly confirm its identity and possession of the red fluorescent protein gene
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 how restriction enzymes are used to create recombinant plasmids

7. 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

8. 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)

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

10. Plasmids as Vectors

11. 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.

12. 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

13. 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))

14. Lab 2A – Digesting pARA-R (cutting the recombinant plasmid)
Purpose: to produce DNA fragments of appropriate size that will confirm the presence of the rfp gene.
Will need to cut recombinant plasmid (construct)
pARA-R – has the rfp gene with promoter sequence (pBAD), an antibiotic resistance gene for ampicillin (ampr) and a regulatory gene (araC) that codes for arabinose activator protein
- Arabinose is a sugar that induces transcription of the rfp gene by working with arabinose activator protein
Will use two restriction enzymes on the construct, liberating the rfp insert from the remainder of the plasmid/vector
BamHI
HindIII

15. Recombinant plasmid (pARA-R)

16. Expected Resulting Digest Fragments
Lab 2A – Digesting pARA-R
Expected Resulting Digest Fragments
BamH I
Hind III
4,495 bp
Hind III
BamH I
807 bp

17. Safety-Lab 2A
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

18. Lab & Aliquoting Guide-Lab 2A
Reagents/Supplies
Aliquot
Storage Temp
Notes
1.4 ml 2.5X Buffer/class (2.5xB)
20ul/group 4o
110 ul of recombinant plasmid (RP)/ class (pARA-R)
10ul/group
-20o
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

19. Completing Lab 2A 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

20. Completing Lab 2A (pg. C-15 in teacher manual)
Label tubes (“R+” & “R-”)
Add 4.0 uL 2.5xB into each
Add 4.0 uL of RP into each tube
Add 2.0 uL RE into R+ tube and mix
Add 2.0 uL dH2O in R- tube 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
* - if time is limited, reducing the digest time to minutes is aaceptable

21. 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

22. Paper Plasmid Activity
Modeling Activity: Choose the appropriate restriction enzyme(s) to use to insert the vasopressin gene into a plasmid-based vector
Recombinant DNA Synthesis Handout (PDF) (PPT) – These may take
several minutes to download