4) Experimental process

kissimmeemisologistΒιοτεχνολογία

14 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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20
th
, April,
2009


Genetically engineered DNA
prepared by transplanting or
splicing genes from one
species into the cells of a
host organism of a different
species. Such DNA becomes
part of the host's genetic
makeup and is replicated.



"The terms recombinant DNA technology, DNA cloning, molecular cloning, or
gene cloning all refer to the same process



Definition: the transfer of a DNA fragment of interest from one organism to a
self
-
replicating genetic element such as a bacterial plasmid (cloning vector).
The DNA of interest can then be propagated in a foreign host cell.



History: around since the 1970s,
researchers have been able construct
recombinant DNA molecules and clone them to generate large amounts of
DNA, such as many multiple copies of a specific gene of interest.

Recombinant DNA technology is frequently employed to
amplify DNA fragments containing genes, an essential step
in their subsequent analysis.


The synthesis of large amounts of an Escherichia
coli (E. coli) protein, called phage shock protein A
was induced when filamentous phage infection o
ccurred






1) Alkaline lysis prep

2) Boiling method

3) Lithium
-
based procedure

1: Chromosomal DNA

2: Plasmid DNA

1) Alkaline lysis prep

2) Boiling method

3) Lithium
-
based procedure

1: Chromosomal DNA

2: Plasmid DNA

Principles of Alkaline lysis prep.

1
.
PCR


2. Agarose gel Electrophoresis


3. Gel Extraction


4. Plasmid Preparation

Today’s schedule: 1 ⇒ 4 ⇒ 2 ⇒ 3



파이펫

별로

정해진

용량

범위

내에서

사용한다

누르다가

걸리는

곳까지가

정확한

용량이므로



눌러서

사용하지

않는다
.

용액을

옮길



천천히

조작한다

용액이

담긴

상태로

파이펫을

거꾸로


세우거나

눕히지

않는다

용액의

위쪽부터

천천히

흡입하며

들어간다
.

** PCR is very sensitive at a extremely small quantity of DNA. So, all reagents should be
stored as aliquots, and tip, tube and water for PCR should be sterilized.

1) Program as follows in PCR machine








2) Prepare reaction mixture as follows







3) Do pipetting gently and run the program of PCR machine, and put the PCR tube in






Cycle step

Temperature

Time

Cycle


number

Initial

denaturation

94℃

5min

1

Denaturation

94℃

30sec

35

Annealing

60℃

30sec

35

Extension

72℃

30sec

35

Final extension

72℃

10sec

1

4℃

hold

Template

1ul

10X

PCR buffer

5ul

Primer 1 (forward)

1ul

Primer 2 (Reverse)

1ul

dNTP

Mixture (2.5
mM

each)

4ul

3
rd

distilled

water

37.75ul

Taq

DNA

polymerase (5units/
ul
)

0.25ul

Template

1ul

Primer 1 (forward)

1ul

Primer 2 (Reverse)

1ul

2X PCR Pre
-
mix

25ul

3
rd

distilled water

22ul

1) Pick a single colony from a freshly streaked bacterial plate and use it to

inoculate LB plus an appropriate antibiotics. Incubate the culture overnight

with shaking. (E. coli strain: JM109, Plasmid: pGEM3)


2) Harvest 3
-
5ml of bacterial culture by centrifugation at 13,000rpm for 30
sec at RT and discard the supernatant.


3) Resuspend the pellet in 250


of Resuspension buffer, vortexing or
pipetting until no clumps of the cell pellet remain.


4) Add 250


of Lysis buffer to resuspended cells. Close tube and gently mix

by inverting the tube several times. Do not vortex and do not exceed 5 min

for lysis time.


5) Add 350


of Neutralization buffer and gently mix by inverting the tube
several times.

6) Centrifuge at 13,000rpm for 10 min at 4℃. While waiting for the
centrifugation, insert a column into collection tube.


7) After centrifugation, transfer supernatant promptly into the column.


8) Centrifuge at 13,000 rpm for 60 sec. Remove the column from collection
tube, discard filtrate in collection tube. And then place the spin column
back in the same collection tube.


9) (Optional) Add 500


of Washing buffer A and centrifuge at 13,000rpm for

60 sec. Remove the column from collection tube, discard filtrate in

collection tube. And then place the spin column back in the same collection

tube.

10) Add 700


of Washing buffer B, centrifuge at 13,000 rpm for 60 sec.
Discard filtrate in the collection tube and place the spin column back in the
same collection tube.


11) Centrifuge at 13,000 rpm for 60 sec to dry the filter membrane.


12) Put the column into a clean and sterile centrifuge tube. Add 20


of
distilled water to the upper reservoir of the column, and let it stand for
1min. Then, centrifuge the tube assembly at 13,000 rpm for 60sec.

1) Agarose gels are covered with TAE buffer solution

2) Prior to loading the samples, the DNA must be mixed with a DNA loading dye
(6X)

Ex) DNA sample 10ul + DNA loading dye (6X) 2ul


3) Load DNA sample into a lane of an agarose gel


4) Running an agarose gel
for about 25 minutes or until the dye travels 1/2
-

to


1/3 of the way down the gel


5) Stain the gel with ethidium bromide (or other DNA staining dye)

Before boiling

After boiling

Template size of PCR: 669
bp

1) To reduce nicking, irradiate the gel for the absolute minimum time possible.
Excise the DNA fragment of interest in a minimal volume of agarose using a
clean scalpel or razor blade. Transfer the gel slice to the weighed micro tube


2) Add 3 volumes BNL Buffer (buffer for gel
-
melting) to 1 volume of gel


3)
Vortex the mixture and incubate at 55℃for 10 minutes or until the gel slice is
completely dissolved.


4) Place one spin column (blue color) in a Collection Tube for each dissolved gel
slice or PCR reaction product.


5) Transfer the dissolved gel mixture or prepared PCR product to the spin column
assembly.


6) Load the sample to the spin column and centrifuge at 13,000rpm for 1min.
Discard the flow
-
through after centrifuging and place the spin column
backin

the same 2 ml collection tube.

7) Add 700
ul

of Washing Buffer to column and centrifuge at 13,000rpm
for1min. Discard the flow
-
through after centrifuging and place the spin
column back in the same 2 ml collection tube.


8) Centrifuge for 1 min at 13,000rpm to dry the spin membrane.


9) Place the spin column to a clean 1.5 ml micro tube. Apply 20ul of the
distilled water directly to the center of the column without touching the
membrane with the pipette tip. Incubate at room temperature for 1 minute.
Centrifuge for 1 minute at 13,000rpm.


10) Discard the spin column and store the micro tube containing the eluted
DNA at
-
20℃.

1.
Three basic steps in PCR reaction: name of the each step and event
in the each step.


2. The similarities and the differences between mini
-
prep and gel
extraction


3. The principle of plasmid DNA isolation from chromosomal DNA in
mini
-
prep of plasmid: each procedure in mini
-
prep and the role of
each buffer


4. Invest the kinds of vectors, and describe its characteristics