Transcript File - South Waksman Club

WSSP-14 Chapter 1
Vectors and Libraries
Today you will start doing
something in the lab called
"Molecular Cloning"
Or
"Genetic Engineering"
Or
"Recombinant DNA Technology"
These techniques will allow you to
study and manipulate individual genes
For many years,
biochemists had tried
to purify genes.
But they were frustrated
because they are hard to
purify.
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?
Genetic Engineering
Nobel Prizes
Paul Berg
Herb Boyer
(Genetech)
Stanley Cohen
Vector containing insert is transformed
into E. coli for amplification and purification
Amplify and Prep
p. 2-1
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
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
p. 1-4
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. 1-4
Transforming plasmids into bacteria
Very inefficient: less than 1/1000 cells are
transformed with the circular plasmid
(linear does not transform)
p. 1-2
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)
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?
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
Plate cells on media with antibiotic
Kills cells without the plasmid
Cloning a DNA fragment
Dead
Cells
Colony
p. 1-2
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
LacZ b-galactosidase, Jacob & Monod
X-gal
Screening for Inserts
p. 1-3
Transform
plasmid into
bacteria
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
Construction and analysis
of a genomic DNA library
Shotgun
sequencing
p. 1-5
Construction and analysis
of a genomic DNA library
Construction and analysis
of a genomic DNA library
Construction and analysis
of a genomic DNA library
Want large clones to span the genomic DNA
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)
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?
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.
mRNA is a processed RNA
transcript (in eukaryotes).
It is intended to be translated
into a protein.
Construction of a
cDNA library
Why are there
blue colonies?
p. 1-6
Construction of a
cDNA library
Construction of a
cDNA library
?
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
Purification of mRNA
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
Synthesis of cDNA from mRNA
p. 1-8
SfiI digestion sites of pTRiplEX2
p. 1-9
Cloning Duckweed cDNA
fragments into the pTriplEX2
polylinker
cDNA Insert
p. 1-10
WSSP-14 Chapter 1B
Plasmid Preps
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
Naming your clones
Your initials
Year
20AV12.14
School #
Clone #
# School
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
Enter the names of the clones into your
school’s Google Docs Clone Report
sheet
The average ON contains 109 cells/ml.
Grown ON on
the bench at RT
Grown ON
shaking at
37°C
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
Fully Grown
Needs to
grow longer
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
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.
2a. Centrifuge the samples
Before
After
Make sure there is a good size pellet
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.
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
3. Resuspend the cell pellet in Soln. I
Resuspend the bacterial pellet in 200 µl of
Solution I by pipeting up and down.
3. Resuspend the cell pellet in Soln. I
Make sure the pellet is fully resuspended!
Not fully
suspended
Fully
suspended
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.
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
4. Add Solution II (cont.)
The cell solution should become clear
Before
After
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
5. Add Solution III (cont.)
Before
Inverting
Inverting
3 times
White precipitate
Inverting
6 times
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.
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.
7. Transfer sup. (DNA) to spin column.
Before
After
8. Centrifuge the spin column
Centrifuge for 1 minute at full speed.
Before
After
8. Centrifuge the spin column (cont.)
Pour the flow-through from the collection tube.
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
9. Wash the column with Wash Buffer
Before
After
Spin
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
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
11. Elute the DNA with EB (cont.)
Centrifuge at full speed for 1 minute.
p. 1-15
12. 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).
p. 1-15