2. Laboratory 3: building a recombinant plasmid
LSSI Alum,
Lindsey Engle
LSSI Alum,
Shawn Hurley
LSSI Alum,
Mary Haus

3. 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

4. Goals of Lab 3
Logistical (students will coordinate procedural steps necessary to):
Synthesize recombinant plasmid (pARA-R), possessing the red fluorescent protein (rfp) gene, ampicillin resistance gene (ampr) and regulatory gene (araC), for future cloning and expression
Educational (students will be able to):
Explain how DNA ligase is used to create a recombinant plasmid
Describe possible recombinant plasmids that form when ligating a restriction digest

5. What are Ligases?
Enzymes that function like molecular glue, bonding two separate pieces of DNA together.
Ligation involves forming covalent bonds in the sugar-phosphate backbone generating a contiguous piece of DNA.
Role in Gene Cloning: When the unpaired bases of two sticky ends come together and form hydrogen bonds, the DNA ligase catalyzes the formation of the covalent bonds between adjacent nucleotides.

6. What are Ligases?

7. 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

8. Recombinant plasmid of interest

9. 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

10. Lab & Aliquoting Guide-Lab 3
Reagents/Supplies
Aliquot
Storage Temp
Notes
30 ul Ligase/class (Lig)
2ul/group
-20o
50 ul 5xB (Ligase B)/ class (5xB)
5ul/group
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

11. 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

12. Completing Lab 3 (pg. B-33 in teacher manual)
***complete paper plasmid activity (see next slide)
Check reagents
Place K+ & A+ tubes into 70oC water bath for 30 min. to inactivate restriction enzymes*
Label LIG tube with group and class period
DIY Water Bath
* - If time is limited, 90oC for 5-10 min. is acceptable

13. 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

14. Completing Lab 3 (continued from previous slide)
Remove K+ and A+ from 70oC 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 dH2O into LIG
Mix reagents in LIG
Place LIG, K+, and A+ in rack
Centrifuge LIG tube
Incubate at room temperature overnight

15. Teacher Resources
Making a recombinant (good, short clip):
Recombinant DNA animation:
Overall process – Good short animation:
Recombinant DNA quizlet:

16. Note on Sequencing Suggested sequences are included in your teacher guides, lessons today were not necessarily presented in this order.
Lab 2 Sequence (pg. B-4)
Session 1 – Review, questions, and discussion
Session 2 – “Clone that Gene” paper plasmid activity and questions
Session 3 – Complete lab 2
Lab 3 Sequence (pg. B-24)
Session 1 – Review and questions
Session 2 – Complete lab 3