Lecture 2: Vectors, cDNA Libraries, & Plasmid

Minipreps
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).
p. 2-1
Cloning Scheme
Digest Ligate Amplify and Prep
p. 2-1
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., pBlueScript
TM
).

Each has a different use, and there are many derivatives of
these basic building blocks. In this course, you will be using
plasmid vectors.
p. 2-1
Plasmids
Circular DNA molecules found in bacteria
Replicated by the hosts 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. 2-1
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
p. 2-2
Transforming plasmids
into bacteria
p. 2-2
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. 2-3
Screening for Inserts
p. 2-3
Plasmid cloning vector
pDONR222
p. 2-4
Cloning a DNA fragment into pDONR222
Lethal
Viable
Transform Transform
p. 2-4
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. 2-5
Construction and analysis
of a genomic DNA library
p. 2-5
Construction of a
cDNA library
p. 2-6
Differences between a genomic and cDNA library
p. 2-7
Genomic Library
Promoters
Introns
Intergenic
Non-expressed genes
cDNA Library
Expressed genes
Transcription start sites
Open reading frames (ORFs)
Splice points

Purification of mRNA
p. 2-8
Collect and grind up animals
in mild denaturing solution
Spin out debris (Tissue,
membranes, etc)
Treat with DNAse
(removes DNA)
Treat with Phenol
(removes protein)
Synthesis of cDNA from mRNA
p. 2-8
Cloning a DNA fragment into pDONR222
Lethal
Viable
Transform Transform
p. 2-4
Preparing Plasmid DNA
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
p. 2-10
Essential components of minipreps
Gentle lysis step to break open the cells and
release the plasmid DNA into solution.
Cell debris and chromosomal DNA of the
bacteria is pelleted during the centrifugation.
Plasmid DNA remains behind in the clear
nonpelleted fraction (the nonpelleted solution left
after centrifugation is known as the
supernatant).
Subsequent steps are then performed on the
supernatant to remove contaminating RNA and
proteins from the plasmid DNA.
p. 2-10
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 37C with
vigorous shaking.
1. Grow the bacteria
p. 2-11
T20AV12.09
Naming your clones
Year Your initials
Group # Clone #
Day
T (Teusday), W (Wednesday), H (Thursday)
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.
p. 2-11
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.

p. 2-11 (fig not shown)
3. Resuspend the cell pellet
Resuspend the bacterial pellet in 250 l of
Buffer PI by vigorous vortexing.
Add 150 ml of PI, 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.

PI contains two essential components: Tris and
EDTA.

Tris is 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. 2-11
4. Add Solution P2
Add 250 l of Solution P2 (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. 2-12
5. Add Solution N3
Add 350 l of Solution N3 (3 M
KOAc, pH 4.8). Mix gently 4-6
times. Do not vortex.
P3 neutralizes cell suspension. A
white precipitate consisting of
aggregated chromosomal DNA,
RNA and cell debris and SDS will
form.
Plasmids will renature

p. 2-12
6. Centrifuge cell debris
Centrifuge for 10 minutes at full
speed in the microcentrifuge.
A white pellet will form on the
bottom and side of the tube after
centrifugation.

p. 2-13
7. Transfer 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.


p. 2-13 (Fig not shown)
8. Centrifuge the spin column
Centrifuge for 1 minute at full
speed, and drain the flow-
through from the collection
tube.


p. 2-13
9. Wash the column with PE
Add 750 ul of Wash buffer PE 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.

p. 2-13
10. Spin the column to remove PE
Place the 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.

p. 2-14 (Figure not shown)
11. Elute the DNA with EB
Place the spin column into an
appropriately labeled 1.7 ml
microcentrifuge tube and add 50 ul of
EB buffer to the column. Centrifuge
at full speed for 1 minute.
Elutes the plasmid DNA from the
column and collects in the
microcentrifuge tube.
p. 2-14
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. 2-14
Other types of vectors
1. Phage Vectors
p. 2-15
2. Phagemids
Have plasmid and phage components
F1 Phage origin of
replication for
making single strand
DNA
p. 2-15
3. Cosmids - Plasmids with phage packaging sites
p. 2-16
4. YACs - Yeast Artificial Chromosomes
p. 2-17
Vector Insert Size
Vector Type Cloned DNA (Kb)
Plasmid 20
Phage 25
Cosmid 45
BAC 300
YAC 1000

p. 2-17