1. Introduction to Vectors In order to study a DNA fragment (e.g., a gene), it needs to be amplified and eventually purified. These tasks are accomplished by cloning the DNA into a vector. A vector is generally a small, circular DNA molecule that replicates inside a bacterium such as Escherichia coli (can be a virus). Ch. 1-1

2. Cloning Scheme DigestLigate Amplify and Prep 1-1

3. Vector Types There are three commonly used types of vectors: 1) plasmid vectors (e.g., pUC plasmids); 2) bacteriophage vectors (e.g., phage ); and 3) phagemid vectors (e.g., pBlueScript TM ). Each has a different use, and there are many derivatives of these basic building blocks. In BRITE, you will be using plasmids (phagemids). Ch. 1-1

4. Plasmids Circular DNA molecules found in bacteria Replicated by the host’s machinery independently of the genome. This is accomplished by a sequence on the plasmid called ori, for origin of replication. Some plasmids are present in E. coli at 200- 500 copies/cell Ch. 1-1

5. Plasmids also contain selectable markers. Genes encoding proteins which provide a selection for rapidly and easily finding bacteria containing the plasmid. Provide resistance to an antibiotic (ampicillin, kanamycin, tetracycline, chloramphenicol, etc.). Thus, bacteria will grow on medium containing these antibiotics only if the bacteria contain a plasmid with the appropriate selectable marker. Plasmid Engineering Ch. 1-2

6. Safety Features Modern cloning plasmids have been engineered so that they are incapable of transfer between bacterial cells Provide a level of biological containment. Naturally occurring plasmids with their associated drug resistance genes are responsible for the recent rise in antibiotic-resistant bacteria plaguing modern medicine. Ch. 1-2

7. Transforming plasmids Into bacteria Ch. 1-2

8. Screening for Inserts 1-3

9. Size of the cDNA insert? cDNA Insert Ch. 1-4

10. Vector Preparation In order to use a vector for cloning, sequencing, etc., it is necessary to isolate the vector in a highly purified form. Routinely done by most labs. Many companies now sell “kits” which provide all the solutions necessary for preparing DNA. Based on similar procedures Ch. 1-4

11. Grow an overnight (ON) culture of the desired bacteria in 2 ml of LB medium containing the appropriate antibiotic for plasmid selection. Incubate the cultures at 37°C with vigorous shaking. Grow the bacteria Ch. 1-6

12. #School 0- Rutgers Univ. 1- Bayonne 2- Bordentown 3- Bridgewater-Raritan 4- Colonia 5- East Brunswick 6- Franklin 7- Hillsborough 8- James Caldwell 9- JFK Memorial 10- JP Stevens 11- Monmouth 12- Montville 13- New Brunswick 14- Pascack Hills 15- Pascack Valley 16- Pingry 17- Rutgers Prep. 18- Watchung Hills 19- West Windsor-Plains. 0AV06-12 Naming your clones School # Your initials Year Clone #

13. 1. Plasmid MiniPreps Obtain your overnight cultures: Bacteria grown in 2 ml of LB medium containing the appropriate antibiotic for plasmid selection. This culture was incubated overnight at 37°C with vigorous shaking. Ch. 1-6

14. 2. Transfer the cells to a tube and centrifuge Transfer 1.5 ml of the culture to a microfuge tube and pellet the cells for 1 minute at full speed (12,000 rpm) in the microcentrifuge. First tap or gently vortex the glass culture tube to resuspend the cells which have settled. The culture can be transferred to the microfuge tube by pouring. Ch. 1-6

15. 2b. Remove the supernatant Remove the growth medium (supernatant or sup) by aspiration or by using the P-1000. Leave the bacterial pellet as dry as possible so that additional solutions are not diluted. Ch. 1-6

16. 3. Resuspend the cell pellet Resuspend the bacterial pellet in 150 µl of Buffer Solution I by vigorous vortexing. Add 150 ml of Solution I, cap the tube, and vortex on the highest setting (pipetman can be used). Look very closely for any undispersed pellet before proceeding to the next step. It is essential that the pellet be completely dispersed. Solution I contains three essential components: Glucose and Tris are used to buffer the pH of the cell suspension. EDTA is a chemical that chelates divalent cations (ions with charges of +2) in the suspension, such as Mg ++. This helps break down the cell membrane and inactivate intracellular enzymes. Ch. 1-6

17. 4. Add Solution II Add 150 µl of Solution II, mix gently 10-15 times. Close the tube tightly and mix the contents by slowly inverting the tubes five times. During this step a viscous bacterial lysate forms (the cells lyse). Do not vortex!! This will shear the DNA and contaminate your DNA preps. Ch. 1-7

18. 5. Add Solution III Add 300 µl of Solution III. Mix gently 10-20 times. Mix by inverting the tubes several times. Do not vortex. A white precipitate consisting of cell debris and SDS will form. Ch. 1-7

19. 6. Centrifuge cell debris Centrifuge for 5 minutes at full speed in the microcentrifuge. A white pellet will form on the bottom and side of the tube after centrifugation. During this centrifugation step, place the necessary number of spin columns into the respective number of 2 ml Collection Tubes and label each appropriately. This is also an ideal time to label 1.7 ml microcentrifuge tubes for use in the final step to collect the miniprep DNA. Ch. 1-7

20. 7. Transfer the sup (DNA) to spin column. Using a P-1000 set at 600ul, transfer the supernatant to the appropriately labeled spin column which has been inserted into the 2 ml microcentrifuge tube. Do not contaminate the spin column with the white precipitate. Ch. 1-8

21. 8. Centrifuge the spin column Centrifuge for 1 minute at full speed, and drain the flow-through from the collection tube. In a single action, remove the spin column from the 2 ml Collection Tube and pour the flow-through, or liquid that passed through the column, into the waste container. Place the spin column back into the 2 ml Collection Tube. Ch. 1-8

22. 9. Wash the column Add 400 ul of Wash buffer to the spin column contained in the 2 ml Collection Tube, centrifuge at full speed for 1 minute, and drain the flowthrough. This buffer helps to further remove any nucleases that may have co-purified with the DNA. Remove the liquid that has passed through the column in the same way as performed in Step 9. Ch. 1-9

23. 10. Spin the column Place the AB spin column in a fresh 1.7 ml microcentrifuge tube (with lid cut off) and centrifuge again for 1 minute at full speed to remove any residual wash solution that might still be in the column. Any residual wash solution must be removed because the ethanol contained in this solution may interfere with further DNA manipulations. It is normal to remove a small amount of liquid from the column at this step, however if a significant amount of solution (50-100 ul or greater) is found in the collection tube, repeat this step. Ch. 1-9

24. 11. Elute the DNA Place the spin column into an appropriately labeled 1.7 ml microcentrifuge tube and add 50 ul of sterile waterto the column. Centrifuge at full speed for 1 minute. This is the final step and elutes or removes the plasmid DNA from the column and back into solution so that it collects in the microcentrifuge tube. This 50 ul of solution contains your plasmid DNA Ch. 1-9

25. Store your DNA Remove the spin column from the labeled 1.7 ml microcentrifuge tube and close the lid on the tube tightly. Store the miniprep DNA in your freezer box (-20C).

26. Plasmid Problem Set: 1. What are vectors used for? 2. What is a polylinker? 3. What is a selectable marker? 4.In preparing double-stranded plasmid DNA preps: a)The first step of the prep is to centrifuge the culture. Do you want to save the supernatant or the pellet? b)Name two functions for Solution II. c)Why does the cell suspension become viscous after adding Solution II ? d)After you add the cell lysis supernatant to the spin column and spin do you want to save the liquid in the bottom of the collection tube or the column? e)After you add the 50 ul of water to the spin column and centrifuge do you want to save the column or the liquid in the bottom of the tube?