Manipulating DNA and Genetic Engineering ... - BIOTECH Project

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Dec 11, 2012 (4 years and 8 months ago)

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BIOTECH Project: Restriction Digests of DNA

University of Arizona





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TEACHER GUIDE: Manipulating DNA and Genetic Engineering

Restriction Digests of DNA


How can we take genes from one organism and connect them with genes from another organism?



Using restriction enzymes, we can cut DNA from each organism and then ligate it

together (e.g. the
ampicillin resistance gene and the luciferase gene into the plasmid backbone).


Why would we want to do this?


This is extremely useful if you want to have an organism make a protein it doesn't normally make (e.g.
bacteria making insuli
n). If we take the human insulin gene, cut it out of the human genome using
restriction enzymes, ligate it into a plasmid with the ampicillin resistance gene, and transform bacteria with
this plasmid, then all the ampicillin resistant bacteria will be mak
ing insulin that can be used to treat
diabetes.



Restriction digest plan

I’m using the restriction enzyme(s):


HindIII




EcoRI

I’m using the buffer color/number:




2




3



Step 1:

Label your tube with your initials and the enzyme you’re using.


St
ep 2:

Pipette 10 µl water into the bottom of your tube.


Now you will visit 3 stations. Please check them off as you visit each station so you don’t have to remember
where you’ve already been. You can visit them in any order.


DNA Station








Done



Add 6 microliters (µl) of DNA to your tube.


Buffer Station
(Why do we add buffer?)





Done



Which buffer are you using?




Add 2 microliters (µl) of buffer to your tube.



Enzyme Station








Done



Which enzyme are you using?




Add 2 microliter
s (µl) of restriction enzyme to your tube.



Why do we use different buffers with different enzymes?


Enzymes are proteins that catalyze reactions. Each reaction and thus each enzyme has a specific
environment where it will work best (e.g. acidic, neutra
l pH, very salty, not salty etc).

BIOTECH Project: Restriction Digests of DNA

University of Arizona





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When you’ve visited all the stations, put your tube into the 37°C heat block.
Why do we put the reactions at
37°C?


R
estriction enzymes are isolated from bacteria. In fact, they serve as a bacterium's immune system by
c
utting up foreign DNA (like viral DNA). The environment of the bacteria is typically 37°C, which means
that these enzymes have evolved to work best at 37°C.


INCUBATE THE TUBES FOR AT LEAST 1 HOUR (can go overnight).

STUDENTS MUST ADD 5 MICROLITERS LOAD
ING DYE TO EACH OF THEIR RESTRICTION
DIGESTS PRIOR TO LOADING THEIR GELS.

















Analyzing Your Restriction Digest Data


Materials

DNA from restriction enzyme digests

molecular weight markers

agarose

Tris
-
acetate/EDTA solution (TAE)

micropipette/
tips

electrophoresis apparatus


Procedure:


1. Get your electrophoresis apparatus. Make sure the comb is in place (one comb near the black electrode) and
that there are stoppers at both ends of the gel space.


2. Pour hot agarose into the gel space un
til it reaches the top of the gel box. Let the agarose harden, which should
take about

10 minutes. Don’t touch/move your gel until it’s hard.
Why not? If the agarose moves while it's hardening,
it will harden unevenly, making it more difficult for th
e DNA to move through evenly.


3. When the agarose gel is hard, take out the stoppers and pour TAE solution over your gel so that is it
completely covered

plus a little more.
What do you think the TAE solution is for? TAE is like saltwater
-

it conducts

electricity,
plus it is a buffer to control for pH changes


4. Gently remove the stoppers and comb.


5. Load your
RESTRICTION DIGEST SAMPLES
into the wells near the
BLACK ELECTRODE
.
Why
near the black electrode?
Be sure to keep track of which sampl
es you loaded in which lanes.
DNA is
negatively charged, so to move the DNA into the gel with electricity, the DNA needs to be loaded on the
negative or black side, it will then move towards the red. If it's loaded near the red electrode, it will
migrate

off the gel into the TBE.


6. Load your
MOLECULAR WEIGHT MARKER
into another well beside your samples. Be sure to keep
track of where you loaded your markers.


7. Run that gel!! Plug the electrodes into your gel box (
red to red, black to black
), being

careful not to bump
your gel too much. Plug the power source into an outlet.
How can you tell your gel is running?

It bubbles at
the electrodes. This is a redox reaction, forming H
2

gas at the black electrode and O
2

gas at the positive
electrode.


Dra
w a picture of your gel and label which samples are where before you add DNA to the gel.


The samples are indistinguishable once they are loaded, so everyone needs to have a drawing of what they
will load where before they load their DNA samples. This dra
wing will be useful during the analysis, once
the gels are stained.

BIOTECH Project: Restriction Digests of DNA

University of Arizona





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Plug in your gel electrophoresis tray, and after
3 minutes
draw another picture.


The blue samples will move to the right (towards the positive red electrode) and separate into light blue

and purple. They move to the positive electrode because they are negatively charged. They separate
because they are different sizes, purple moves faster and farther because it is smaller (lower molecular
weight).


The DNA cannot be observed until the ge
l is stained because DNA is colorless. We add blue coloring to
the DNA samples so you can see the samples as you are loading them.


Run the gels until the purple travels 2/3 of the way across the gel (about 25
-
30 minutes).


Once the purple dye has migrat
ed approximately 2/3 of the gel, turn off the power and carefully remove the gel.
The gel is very fragile, take care to not break it. You can remove the tray that you poured agarose on to and
gently slide the gel into the staining tray. At this point you c
annot see the DNA, what can you see and how do
the four different lanes compare?


The four lanes should all be the same. You should see two dyes in each lane, purple that migrated the
furthest and a blue that is closer to the wells. The purples should hav
e migrated the same distance from
the wells, as the blues should have migrated the same distance from the wells.


Once you have placed your gel into the staining tray bring it to the staining station. Completely cover the gel
with methylene blue and cover

try with saran wrap. Stain overnight.


Next day
--
Viewing the gel: Pour the methylene blue back into the bottle and carefully place the gel onto a white
light box. The gel is very fragile so take care to not break it. Draw a picture of your stained gel:


I used restriction enzyme:




How many cuts did your restriction enzyme make?


The number of cuts will be one less than the number of DNA fragments (i.e. if you cut a string once,
you'll have two pieces, twice and you'll have three pieces, etc). The sma
ller the pieces of DNA, the harder
it will be to see them, so students may differ on their answers depending on how many pieces they can see
from their data.



Use the molecular weight markers loaded in your gel along side your restriction digest reactions

to plot a graph
on semi
-
log paper (distance traveled on the X
-
axis and DNA size on the Y
-
axis). Use your plot to serve as a
standard to determine the size of DNA fragments your digest reactions yielded.



Molecular weight markers (ladder):

BIOTECH Project: Restriction Digests of DNA

University of Arizona





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Recipes an
d Background Information


• 6X loading dye


6 ml 50% glycerol

1 ml 2% bromophenol blue

1 ml 2% xylene cyanol

2 ml distilled water


• Tris
-
borate
-
EDTA buffer for dye electrophoresis


5X stock (per liter)

54 g Tris base

27.5 g boric acid

20 ml 0.5M EDTA (pH

8.0)

Add distilled water to make total volume 1 liter. Dilute to make 1X working solution (200 ml stock, 800 ml
distilled water).


• 0.8% Agarose gel for dye electrophoresis


In 250 ml Pyrex bottle, combine:


125 ml 1X TBE

1 g agarose


Microwave uncover
ed to melt agarose, be careful not to boil over on microwave or your hand. Cover loosely
and store at room temperature.


• 0.025% Methylene blue stain


Make 1% methylene blue stock: 99 ml water and 1 g methylene blue powder.


10 ml 1% methylene blue stock

390 ml distilled water