1. WSSP-14 Chapter 1
Vectors and Libraries

2. Today you will start doing something in the lab called
"Molecular Cloning" Or
"Genetic Engineering"
"Recombinant DNA Technology"
These techniques will allow you to study and manipulate individual genes

3. For many years, biochemists had tried to purify genes.
But they were frustrated because they are hard to purify.

4. Because genes are composed of
A’s, C’s, G’s, and T’s, they all pretty much are chemically alike.
Also genes are parts of chromosomes. Chromosomes break easily and randomly, often in the middle of genes.
So how did scientists eventually purify individual genes?

5. Genetic Engineering Nobel Prizes Paul Berg Herb Boyer (Genetech)
Stanley Cohen

6. Vector containing insert is transformed
into E. coli for amplification and purification
Amplify and Prep
p. 2-1 6

7. 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).
p. 1-1

8. 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 copies/cell
p. 1-4 8

9. Plasmid Engineering p. 1-4 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.
p. 1-4 9

10. Transforming plasmids into bacteria
Very inefficient: less than 1/1000 cells are transformed with the circular plasmid
(linear does not transform)
p. 1-2 10

11. Transforming plasmids into bacteria
Need to treat cells with Ca++ to transform plasmid
Normal Cell
(negatively charged membrane)
Plasmid
Ca++
Cell treated with CaCl2
(more postively charged membrane)

12. Transforming plasmids into bacteria
Very inefficient: less than 1/1000 cells are transformed with the plasmid
How do you identify the few cells with the plasmid?

13. Plasmid Engineering 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.
p. 2-2

14. Plate cells on media with antibiotic
Kills cells without the plasmid

15. Cloning a DNA fragment
Dead
Cells
Colony
p. 1-2 15

16. 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.
p. 1-3 16

17. LacZ b-galactosidase, Jacob & Monod
X-gal

18. Screening for Inserts
p. 1-3 18

19. Transform plasmid into bacteria

20. DNA Libraries
DNA library - a random collection of DNA fragments from an organism cloned into a vector
Ideally contains at least one copy of every DNA sequence.
Easily maintained in the laboratory
Can be manipulated in various ways to facilitate the isolation of a DNA fragment of interest to a scientist.
Numerous types of libraries exist for various organisms - Genomic and cDNA.
p. 1-5

21. Construction and analysis
of a genomic DNA library
Shotgun
sequencing
p. 1-5

22. Construction and analysis of a genomic DNA library22

23. Construction and analysis of a genomic DNA library23

24. Construction and analysis of a genomic DNA library24

25. Want large clones to span the genomic DNA

26. Sequencing the human genome cost $3 billion.
Efforts are being made to cut the cost of sequencing a specific human genome to $1,000 (or less)

27. We are not sequencing a genomic DNA library.
We are sequencing a cDNA library
What's that and what is the difference between the two?

28. A cDNA is a copy of RNA (usually mRNA) in the form of DNA.
We are not sequencing a genomic DNA library.
We are sequencing a cDNA library
What's that and what is the difference between the two?
A cDNA is a copy of RNA (usually mRNA) in the form of DNA.

29. mRNA is a processed RNA transcript (in eukaryotes).
It is intended to be translated into a protein.

30. Construction of a
cDNA library
Why are there blue colonies?
p. 1-6

31. Construction of a
cDNA library 31

32. Construction of a
cDNA library ? 32

33. Differences between a genomic and cDNA library
Genomic Library
Promoters
Introns
Intergenic
Non-expressed genes
cDNA Library
Expressed genes
Transcription start sites
Open reading frames (ORFs)
Splice points
p.1-7

34. Purification of mRNA p. 1-8
Collect and grind up plants in mild denaturing solution
Spin out debris (Tissue, membranes, etc)
Treat with DNAse (removes DNA)
Treat with Phenol (removes protein)
p. 1-8

35. Synthesis of cDNA from mRNA
p. 1-8

36. SfiI digestion sites of pTRiplEX2

37. Cloning Duckweed cDNA fragments into the pTriplEX2 polylinker
Look at vector first,
Where are all the the sites that you know about?
cDNA Insert
p. 1-10

38. WSSP-14 Chapter 1B
Plasmid Preps

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

40. 20AV12.14 Naming your clones Your initials Year School # Clone #
01. Bayonne HS, NJ
02. Bridgewater HS, NJ
04. East Brunswick HS, NJ
05. High Point HS, NJ
06. Hillsborough HS, NJ
07. James Caldwell HS, NJ
09. JP Stevens HS, NJ
11. Montville HS, NJ
13. Pascack Hills HS, NJ
14. Pascack Valley HS, NJ
15. Rutgers Prep., NJ
16. Somerville HS, NJ
17. The Pingry School, NJ
18. Watchung Hills HS, NJ
19. West Windsor-Plains. HSS, NJ
38. Hackettstown, NJ
47. Fairlawn NH, NJ
49. Piscataway, NJ
50. The Frisch School, NJ
70. Old Bridge HS, NJ
93. The Peddie School, NJ
94. Academy of Edison, NJ
95. Acad. Of Enrichment & Adv., NJ
96. Holmdel HS, NJ
97. Robbinsville HS, NJ
98. Union City HS, NJ
103. The Hun School, NJ
104. Elmwood Park Memorital, NJ
105 North Brunswick, NJ
# School
34. Science & Math Acad. MD
35. Walter Johnson, MD
59. Col. Zadok Magruder HS, MD
62. Winston Churchill, MD
81. South River HS, MD
82. Southern HS, MD
87. Kent Co HS IBALC, MD
88. Randallstown HS, MD
89. Gilman School, MD
100. Cristo Rey Jesuit HS, MD
101. Pikesville HS, MD
102. New Town HS, MD
# School
65. Dougherty HS, CA
66. Modesto HS, CA
67. Tracy HS, CA
68. Waipuhu HS, HI
90. Granada High School, CA
91. Amador Valley High School, CA
Your initials
Year
20AV12.14
School #
Clone #

41. Enter the names of the clones into your school’s Google Docs Clone Report sheet

42. The average ON contains 109 cells/ml.

43. Grown ON shaking at 37°C
Grown ON on the bench at RT

44. One way to tell if your ON is fully grown is to see if you can see writing if you hold the tube up to your notes
Needs to grow longer
Fully Grown

45. 2a. 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.
(Follow steps in Lab 6)
p. 1-12

46. 2a. Centrifuge the samples
Balance the tubes in the centrifuge
Pellet the cells for 1 minute at full speed (10,000-14,000 rpm) in the microcentrifuge.

47. 2a. Centrifuge the samples
Before
After
Make sure there is a good size pellet

48. 2b. Remove the supernatant
Remove the growth medium (supernatant) by pouring out into a waste cup.
Leave the bacterial pellet as dry as possible so that additional solutions are not diluted.

49. 3a. Resuspend the cell pellet
Resuspend the bacterial pellet in 200 µl of Solution I by pipeting up and down.
Add 200 l 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, Tris and EDTA.
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.
p. 1-12

50. 3. Resuspend the cell pellet in Soln. I
Resuspend the bacterial pellet in 200 µl of Solution I by pipeting up and down.

51. 3. Resuspend the cell pellet in Soln. I
Make sure the pellet is fully resuspended!
Not fully
suspended
Fully
suspended

52. 3b. Store the suspended the cell pellet in you school’s box at -20°C
NEW CHANGE IN PROTOCOL!!
3b. Store the suspended the cell pellet in you school’s box at -20°C
It may take more than two weeks to perform the PCR and run the gel before you are ready to perform the miniprep.
If the bacterial culture is left in the refridgerator then during this time many of the cells will die and the plasmid yeilds will go down.

53. 4. Add Solution II
Add 200 µl of Solution II (0.2 N NaOH, 1%SDS), mix gently 4-6 times. Do not vortex!! This will shear the DNA and contaminate your DNA preps.
Denatures protein, DNA, RNA, membranes. During this step a viscous bacterial lysate forms (the cells lyse).
p. 1-13

54. The cell solution should become clear
4. Add Solution II (cont.)
The cell solution should become clear
Before
After

55. 5. Add Solution III
Add 400 µl of Solution III (3 M KOAc, pH 4.8). Mix gently 4-6 times. Do not vortex.
Solution III neutralizes cell suspension. A white precipitate consisting of aggregated chromosomal DNA, RNA and cell debris and SDS will form.
Plasmids will renature
p. 1-13

56. 5. Add Solution III (cont.)
Before Inverting
Inverting
3 times
Inverting
6 times
White precipitate

57. 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.

58. 7. Transfer sup. (DNA) to spin column.
Pour the supernatant to the appropriately labeled spin column which has been inserted into the 2 ml microcentrifuge tube.

59. 7. Transfer sup. (DNA) to spin column.
Before
After

60. 8. Centrifuge the spin column
Centrifuge for 1 minute at full speed.
Before
After

61. 8. Centrifuge the spin column (cont.)
Pour the flow-through from the collection tube.

62. 9. Wash the column with Wash Buffer
Add 400 l of Wash buffer to the spin column contained in the 2 ml Collection Tube, centrifuge at full speed for 1 minute, and drain the flow through.
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 8.
p. 1-14

63. 9. Wash the column with Wash Buffer
Before
After
Spin

64. 10. Spin the column a second time to remove all the Wash buffer
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.
p. 1-15

65. 11. Elute the DNA with EB
Place the spin column into an appropriately labeled 1.7 ml microcentrifuge tube and add 60 ul of EB buffer to the column. Elutes the plasmid DNA from the column and collects in the microcentrifuge tube.
p. 1-15

66. 11. Elute the DNA with EB (cont.)
Centrifuge at full speed for 1 minute.
p. 1-15

67. 12. Store your DNA Store the miniprep DNA in your freezer box (-20C).
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).
p. 1-15