Transcript Introduction to Vectors - Pascack Valley Regional School District

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
Cloning Scheme
Digest
Ligate
Amplify and Prep
1-1
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., pBlueScriptTM).
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
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 200500 copies/cell
Ch. 1-1
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.
Ch. 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.
Ch. 1-2
Transforming plasmids
Into bacteria
Ch. 1-2
Screening for Inserts
1-3
Size of the
cDNA insert?
cDNA Insert
Ch. 1-4
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
Grow the bacteria
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.
Ch. 1-6
Naming your clones
School #
Year
0AV06-12
Your initials
Clone #
#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.
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
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
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
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
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
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
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
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
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
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
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
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
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).
Plasmid Problem Set:
1.
2.
3.
4.
What are vectors used for?
What is a polylinker?
What is a selectable marker?
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?