2. LABORATORY 2 & 3: HOW DO YOU BEGIN TO CLONE A GENE? LSSI Alum, Mary Haus

3. Pre-Assignment http://www.dnaftb.org/34/animation.html 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 https://www.youtube.com/watch? v=nfC689ElUVk **Please be aware, sticky ends are not identified correctly, but overall process is.

4. 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.

5. Plasmid Features: Ori (replicating/cloning) Promoter Antibiotic resistance Restriction sites

6. 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.

7. 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.

8. 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

9. Lab 2 – Creating the Digest

10. Restriction Digest Fragments 4,706 bp BamH IHind III 806 bp BamH IHind III 376 bp BamH IHind III BamH IHind III 4,496 bp Lab 2 – Creating the Digest

11. www.amgenbiotechexperience.com 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

12. www.amgenbiotechexperience.com Lab & Aliquoting Guide-Lab 2 Reagents/SuppliesAliquotStorage TempNotes 1.4 ml 2.5X Buffer/class (2.5xB)20ul/group4o4o 110 ul of pKAN/class (pKAN)10ul/group-20 o 110 ul of pARA/ class (pARA)10ul/group-20 o 65 ul of Restriction Enzyme/class (RE) 5ul/group-20 o 12mL of DI water/ kit (dH20)1ml/groupRTKeep this for all Labs Equipment/Supplies 10 Student boxes with the following: 1 p20 micropipette 1 microfuge rack 1 p200 micropipette 1 bag of microfuge tubes 1 p1000 micropipette 1 bag of microfuge tubes 1 waste and 1 box of refillable tips (2 ul-200 ul) 1 ice bucket 4 Mini centrifuges 1 Water bath 2 Floating racks

13. 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

14. Completing Lab 2 (pg. B-15 in teacher manual) Label tubesAdd 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 dH 2 O in K- and A- tubes and mix Centrifuge all tubes Put all tubes in floating rack and set in 37 o C water bath for 60 mins. Remove and place tubes into freezer overnight

15. Teacher Video Resources Mixing two solutions video: https://www.amgenbiotechexperience. com/curriculum/curriculum- resources/mixing-two-solutions https://www.amgenbiotechexperience. com/curriculum/curriculum- resources/mixing-two-solutions Digestion video (different digest, but good techniques): https://www.youtube.com/watch?v=Gs Wo8dCivWs https://www.youtube.com/watch?v=Gs Wo8dCivWs How restriction enzymes work (good, short): https://www.youtube.com/watch?v=lW XryzgRces 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 https://www.youtube.com/watch?v=4 wbStjWEM8A Fun one from MIT. Covers whole process 1 st half good for Labs 2&3: https://www.youtube.com/watch?v=nf C689ElUVk https://www.youtube.com/watch?v=nf C689ElUVk ***remember, sticky end issue here

16. What are Ligases? Definition: Enzymes that catalyze the formation of covalent bonds in the sugar phosphate backbone. Ligation is basically the reaction that joins two pieces of DNA together. 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.

17. 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

18. Recombinant plasmid of interest

19. www.amgenbiotechexperience.com 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

20. www.amgenbiotechexperience.com Lab & Aliquoting Guide-Lab 3 Reagents/SuppliesAliquotStorage TempNotes 30 ul Ligase/class (Lig)2ul/group -20 o 50 ul 5xB (Ligase B)/ class (5xB)5ul/group -20 o Equipment/Supplies 10 Student boxes with the following: 1 p20 micropipette 1 microfuge rack 1 p200 micropipette 1 bag of microfuge tubes 1 p1000 micropipette 1 bag of microfuge tubes 1 waste and 1 box of refillable tips (2 ul-200 ul) 1 ice bucket 4 Mini centrifuges 1 Water bath 2 Floating racks

21. 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

22. Completing Lab 3 (pg. B-33 in teacher manual) ***complete paper plasmid activity (see next slide) DIY Water Bath Check reagents Place K+ and A+ into 70 o C water bath for 30 mins Label LIG tube with group and class period

23. Modeling Activity: Choose the appropriate restriction enzyme to use to insert the insulin gene Paper Plasmid Activity (starts on Pg. 43 in student manual)

24. 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 dH 2 0 into LIGMix reagents in LIG Centrifuge LIG tube Place LIG, K+, and A+ in rack Incubate at room temperature overnight

25. Teacher Resources Making a recombinant (good, short clip): https://www.dnalc.org/view/15476- Mechanism-of-Recombination-3D- animation-with-with-basic- narration.html https://www.dnalc.org/view/15476- Mechanism-of-Recombination-3D- animation-with-with-basic- narration.html Recombinant DNA animation: http://www.bioteach.ubc.ca/Teachin gResources/Applications/GMOpkgJKl oseGLampard2.swf 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-working- with-dna/ http://www.pbslearningmedia.org/re source/biot11.sci.life.gen.genengdna /genetic-engineering-and-working- with-dna/ Recombinant DNA quizlet: https://quizlet.com/7435136/recomb inant-dna-technology-flash-cards/ https://quizlet.com/7435136/recomb inant-dna-technology-flash-cards/

26. 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 3 Lab 3 Sequence (p B-24) Session 1 – Review and questions Session 2 – Complete lab 3