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14 Δεκ 2012 (πριν από 4 χρόνια και 10 μήνες)

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WSSP
-
09 Chapter 1

Vectors and Plasmid Preps


Today you will start doing
something in the lab called


"
Molecular Cloning
"

O
r

"
Genetic Engineering
"

O
r

"
Recombinant DNA Technology
"

These techniques will allow you to
study and manipulate
individual

genes

A
-

a chromosome

B
-

a protein

C
-

part of a chromosome that is
transcribed

D
-

a section of DNA that codes for
a protein

E
-

is made of RNA

What is a gene?

For many years,
biochemists had tried

to purify genes.


But they were frustrated
because they are hard to
purify.

Why are genes hard to purify?


a.
Because they
'r
e tiny

b.
Because there are too few of them

c.
Because one piece of DNA looks
like any other

d. Because they are hard to separate
from proteins

e. Because they are too long

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?

Paul Berg

Herb Boyer

(Genetech)

Stanley Cohen

Genetic Engineering

Nobel Prizes

Cloning Scheme

Digest

Ligate

Amplify and Prep

p. 1
-
1

Wolffia

DNA

But why use vectors?

A
-

because passenger DNA won
't

replicate by itself

B
-

because they
'r
e inexpensive

C
-

because they
'r
e circular

D
-

because they
'r
e small

E
-

because passenger DNA is too big
to get into cells by itself

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
-
1

The most
common
bacterial
plasmids
are
members of
the pUC
series

J. Messing

Director, Waksman Institute

Transform
plasmid into
bacteria

Why a maximum of only one
molecule per cell?

A
-

Otherwise the cell will get too crowded

B
-

A cell can
't

replicate two plasmids

C
-

Cells don
't

like to have more than one
plasmid in them

D
-

It will be too confusing to have more
than one plasmid in a cell

E
-

Why not?

How do you make sure that a
maximum of only one plasmid gets
into any cell?


A
-

Make it unlikely that any one cell gets a
plasmid

B
-

Carefully micropipet only one plasmid
per cell

C
-

You throw away the cells that have two
or more plasmids

D
-

Stop the second plasmid going in after
detecting the first

If you make it unlikely that any one cell
gets a plasmid how do you find the
bacteria with the plasmids?




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. 1
-
2

Transforming plasmids

into bacteria

p. 1
-
2

Clicker Question #1: What is the most likely
explanation if the plating of the ligation mix of the
cDNA library transformation looked like this?

A) The ligation did not work

B) The cells were not competent

C) The DNA was not added to the cells

D) The plates did not have chlorampenicol

E) None of the above


(Lawn of cells)

Clicker Question #2: What is the most likely
explanation if the plating of the ligation mix of the
cDNA library transformation looked like this?

A) The ligation did not work

B) The cells were not competent

C) The vector was degraded

D) A, B or C

E) None of the above


(No cells)

p. 1
-
2

A) The ligation did not work

B) The cells were not competent

C) The vector was degraded

(No cells)

Plasmid cloning vector
pDNR
-
Lib


The cDNA insert is cloned into
the
Sfi
I sites

p. 1
-
4

cDNA Insert

MCS A

MCS B

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. 1
-
11

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. 1
-
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.

1. Grow the bacteria

p. 1
-
11

Clicker Question #4: Why is it very important that
you change toothpicks after each colony you pick to
make an ON?

A)
Toothpicks are cheap.


B)
Cells from one colony would be mixed in an ON
with cells from another colony.


C)
The liquid in the culture tube would contaminate
the plate.


D)
It is easier to count how many clones you have
done.


E)
It is not important to change toothpicks

Clicker Question #4: Which of the following colonies
would be best to pick for making an ON?

A)

B)

C)

D)

E)

20AV12.09

Naming your clones

Year

Your initials

School #

Clone #

School #

1. Bayonne

3. Colonia

4. East Brunswick

5. High Point

6. Hillsborough

7. James Caldwell

8. JFK Memorial

9. JP Stevens

10. Monmouth

11. Montville

12. New Brunswick

13. Pascack Hills

14. Pascack Valley

15. Rutgers Prep.

16. Somerville

17. The Pingry School

18. Watchung Hills

19. West Windsor
-
Plains.

20. Rutgers University

21. Liberty

24. Lynbrook

28. Roland Park Country

29. Archbishop Curley

30. Largo

31. DuVal

32. Great Mills

33. McDonogh

34. Science & Math. Acad.

35. Walter Johnson

36. North County

37. Thurgood Marshall

38. Hackettstown

40. Bordentown


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.

p. 1
-
11

(Follow steps in Lab 4
-
1)

3. 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. 1
-
12

4. Resuspend the cell pellet

Resuspend the bacterial pellet in 200 µl of
Solution I by vigorous vortexing
.


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
-
11

5. 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
-
12

6. 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
-
12

7. 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.


p. 1
-
13

8. Transfer sup. (DNA) to spin column.


Using a P
-
1000 set at 800

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獵灥牮慴慮琠瑯s瑨攠t灰牯灲楡p敬e慢敬e搠d灩渠
column which has been inserted into the 2 ml
microcentrifuge tube.





p. 1
-
13

9. Centrifuge the spin column


Centrifuge for 1 minute at
full speed, and drain the
flow
-
through from the
collection tube.





p. 1
-
13

10. Wash the column with Wash Buffer


Add 400

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瑨t 獰楮⁣潬o浮m捯湴慩湥搠楮i瑨t
㈠浬2䍯汬散瑩潮o呵扥T 捥c瑲楦畧攠
慴a晵f氠獰敥搠景爠ㄠ浩湵瑥1 慮搠
摲慩渠瑨t 晬f眠瑨牯畧t
.



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

11. Spin the column to remove Wash
buffer


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. 1
-
14

12. 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.

Centrifuge at full speed for 1
minute.


Elutes the plasmid DNA from the
column and collects in the
microcentrifuge tube.

p. 1
-
14

14. Store your DNA

p. 1
-
15

Remove the spin column from the labeled 1.7 ml microcentrifuge
tube and close the lid on the tube tightly.


A) Transfer 10 ul to a labeled tube to use as the stock for
sequencing. Place it in a storage box with your school
number and your initials and “Seq” on it.

B) The remaining stock will be used for your digests and PCR.
Store it in a box with your school number and your initials
and “Digest” on it.

Store the miniprep DNA in your freezer box (
-
20C).


Enter the name and location of the plasmid in the
spread sheet

Enter the clone
name and prep
date in the clone
storage sheet.


Please also enter
this information
into the electronic
version

Book keeping!!

Clicker Question #5: Which of the following is the
most serious

error in the miniprep procedure?

A)
Not fully resuspending the cells in Solution #1.


B)
Vortexing after Solution #3 is added.


C)
Transferring some of the precipitate onto the
column.


D)
Washing the column with Elution Buffer (EB)
instead of Wash (W).


E)
Eluting with 100 ul Elution Buffer (EB) instead of
50 ul.

Clicker Question #6: After what step is the best
place to stop the miniprep procedure if the class time
runs out?

A)
After resuspending the cells in Solution #1.


B)
After Solution #3 is added.


C)
After spinning out the precipitate


D)
After transferring the supernatant onto the
column.


E)
After washing the column with Wash Buffer.



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

Most sequencing has been done by
the whole genome shotgun method,
pioneered by Craig Venter

Construction and analysis

of a genomic DNA library

p. 1
-
5

Shotgun

sequencing

Want large clones to span the genomic DNA

YACs
-

Y
east
A
rtificial
C
hromosomes

p. 1
-
18

BACs
-

B
acterial
A
rtificial
C
hromosomes

Insert (150
-
350 kb)

Bacterial F episome

Vector Insert Size

Vector Type

Cloned DNA (Kb)

Plasmid

20

Phage

25

Cosmid

45

BAC

300

YAC

1000


p. 1
-
18

What would happen if there
are many long repetitive
sequences in a genome?

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?

A cDNA library is made from a
collection of cDNA
's
.

What's a cDNA?

What's a cDNA?


A
-

a
"cut"

DNA that comes from a
restriction digest

B
-

a
"cloned"

DNA that comes from a
passenger

C
-

a
"complementary"

DNA that is
complementary to mRNA

D
-

a
"closed"

DNA that is circular

E
-

a
"cute"

DNA that has a pretty
sequence

A cDNA is a copy of RNA
(usually
mRNA
) in DNA.

What's a mRNA?

A
-

a
"m
essenger" RNA that is
intended for translation

B
-

a
"m
ajor" RNA that is present in
large amounts

C
-

a
"m
issing
"

RNA that is present in
low amounts

D
-

a
"m
issense
"

RNA that can
't

be
translated into a protein

E
-

a
"m
obile
"

RNA that has moved
from cytoplasm to the nucleus

mRNA is a
processed

RNA
transcript(in eukaryotes).


It is intended to be translated
into a protein.

Construction of a

cDNA library

p. 1
-
6

Differences between a genomic and cDNA library

p.1
-
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. 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)

Synthesis of cDNA from mRNA

p. 1
-
8

Sfi
I digestion sites of pDNR
-
Lib

p. 1
-
9

p. 1
-
10

Cloning
W.a.

cDNA fragments into
the pDNR
-
Lib polylinker