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Ebru AKHARMAN
142204026
26.03.2018
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molecules migrate towards the positive end of the 6) Store the amplified DNA at 4 °C for short term
field, which contains positively-charged agarose gel. storage or at -20 °C for long term storage.
The gel is a cross-linked matrix network that serves as 7) Visualise the 2 μl of the amplified DNA via agarose
a three-dimensional mesh or screen to attract gel electrophoresis (This will be done in the next lab
negatively charged DNA molecules. Those molecules period).
are pulled to the positive end of the gel base by
currents, but encounter resistance from the agarose,
which then isolates DNA molecules. Large molecules MATERIAL AND METHOD FOR
move slower than small molecules. Because of this, it RESTRICTION ENZYME DIGESTION:
is expected that small molecules will be ahead of 39 µl 𝑑𝐻2 𝑂
large molecules. 5 µl Buffer B 10X (This buffer is common for
MATERIAL AND METHOD FOR PCR: EcoRI and HindIII)
10 µl 𝑑𝐻2 𝑂 2 µl Puc19 or Pac3,5(PCR product / 3500 bp /
2,5 µl PCR buffer ( 10 x ) 2 µg)
𝑀𝑔𝐶𝑙2 ( 1.5, 2, 3, 4, 5 µl ) 2 µl EcoII and HindIII( 4U / µl / The diluted
2 µl Forward Primer enzymes are used.)
2 µl Reverse Primer
2 µl Template DNA 1) Mix all the components listed above in a given
2 µl Taq DNA Polymerase order in an eppendorf tube but Puc19 and Pac3,5
2,5 µl dNTPs must be added different eppendorfs (This is
important in order to correct errors that will occur
1) Combine the following materials, except Taq DNA during the experiment.)
polymerase, in a 0.5 ml eppendorf tube (on ice). 2) The tubes are incubated at 37 °C for 90 min. (Most
Prepare also a control reaction with no template DNA restriction enzymes show maximum activity at 37°C.
and an additional 2 μl sterile water instead of For incubations greater than 1 hour with high
template DNA. Groups 1 to 5 use the amount of temperature enzymes, cover the reactions with a
𝑀𝑔𝐶𝑙2 as specified (1.5, 2, 3, 4, 5) and the other drop of mineral oil to prevent evaporation.
groups use 2 microliters. Generally, the incubation temperature for the
2) The tubes are placed in a thermal cycler preheated enzyme reflects the growth temperature of the
to 94 °C for 5 minutes.( For first denaturation step.) bacterial strain from which it is derived.)
3) Taq DNA polymerase is added as a last component 3) 1 μl RNase DNase-free (1/2 diluted) is added and
after the first denaturation step at 94 °C for 5 incubate the tubes at 37 °C for additional 15 min.
minutes.( Otherwise, high temperature disrupts the (Puc19 have RNAs and the RNAs can make pollution
enzyme structure.) so RNAs are digested with RNase.)
4) Perform the following temperature cyles for 30 4) This mixture is centrifuged in 11.000 rpm. Then,
times: ( For denaturation.) the supernatant is removed. 2 μl sodium acetate 3 M
94 °C 60 seconds (pH 5.2) and 125 μl of 100 % ethanol are added.( The
64 °C 60 seconds / 72 °C 210 seconds enzymes prefer acetate to chloride anions. The
5) Perform a final extension step at 72 °C for 10 ethanol-precipitate is maked to remove RNA or
salts.)
minutes.
5) The tubes are waited for -80 °C for 30 min.
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6) Centrifuge in a microfuge (+4 °C) at 14.000 rpm for Preparation of 1 % agarose gel:
15 min. 1) To prepare 30 ml of a 1 % agarose solution,
7) The supernatant is removed and 50 μl of 70 % measure 0.3 g agarose into a glass flask.
ethanol is added. 2) Microwave for 1-3 min (until the agarose is
completely dissolved).
8) Centrifuge in a microfuge (+4 oC) at 14.000 rpm
3) Let agarose solution cool down (~ 50-55 °C) for
for 15 min. about 5 min
9) Remove the ethanol and dry the pellet in a 4) Add 3 μL of 20,000x RedSafe Nucleic Acid Staining
centrifugal evaporator for 10-20 min. Solution to the agarose solution. Swirl the flask gently
10) Resuspend the pellet in 10 μl dH20. to mix the solution and avoid forming bubbles.(Pour
slowly to avoid bubbles which will disrupt the gel.
11) Add 2 μl of 6x gel loading dye. Any bubbles can be pushed away from the well
12) Load the digested DNA sample in a 1 % agarose comb or towards the sides/edges of the gel with a
gel and electrophorese at 80 volts until dye markers pipette tip.)
have migrated an appropriate distance (about 1 5) Place newly poured gel at 4 °C for 10-15 minutes
hour). OR let sit at room temperature for 20- 30 minutes,
until it has completely solidified. (If you are in a
13)Apply the "DNA Isolation from Agarose Gel" hurry, the gel can also be set more quickly if you
protocol. place the gel tray at 4°C earlier, so that it is already
cold when the gel is poured into it.)
MATERIAL AND METHOD FOR 6) To run, gently remove the comb, place tray in the
electrophoresis chamber, and cover (just until wells
AGAROSE GEL ELECTROPHORESIS are submerged) with electrophoresis buffer (the
AND DNA ISOLATION FROM same buffer used to prepare the agarose).
AGAROSE GEL: 7) To prepare samples for electrophoresis, add 1 μl of
6x gel loading dye for every 5 μl of DNA solution. Mix
well. Load 5-12 μl of DNA per well (for minigel).
Agarose (electrophoresis grade) 8) Electrophorese at 100 volts until dye markers have
50x TAE migrated an appropriate distance, depending on the
size of DNA to be visualized.
6x Loading dye
9) Visualise and analyze the gel under UV light using a
RedSafe Nucleic Acid Staining Solution transilluminator. DNA can be visualised under short
(20.000X) wave UV light if the DNA will not be used further; or
with a long-wave UV light if the DNA is to be cut out
and purified.
50x TAE Stock Solution:
242 g Tris base DNA Isolation from Agarose Gel:
57.1 ml glacial acetic acid
37.2 g Na2EDTA.2H2O 1.5 ml microcentrifuge tubes
H2O to 1 l. Disposable pipette tips
pH ̴ 8.5 Scalpel
6x Loading Dye:
Bromophenol Blue (BPB) 0.15 % 11) Excise the DNA fragment from the agarose gel
Sucrose/glycerol 30 % with a clean, sharp scalpel and tranfer it into a
microcentrifuge tube. (Expose the gel to UV light as
short as possible. Use the longest UV wave length
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that is allowed by the gel documentation system. and place the column back into the same collection
Prolonged exposure and short wave lengths can tube.
damage the DNA. Wear gloves and a face mask to 18) Repeat previous washing step to minimize
protect your skin and eyes from UV ligth.) chaotropic salt carry-over and low A260/A230.
12) Weight the gel slice. For each 100 mg of agarose (Carry-over of GuSCN (guanidinium thiocyanate) can
gel add 200 μl of Buffer NTI buffer. lower the A260/A230 ratio from its ideal value of
13) Incubate sample at 50 °C for 5-10 minutes (or >2.0 to below 1.5 or even 1.0.)
until the gel slice has completely dissolved). To help 19) Centrifuge for 1 min 1t 11,000 x g to remove
dissolve gel, mix by vortexing the tube every 2–3 min Buffer NT3 completely. Make sure the spin column
during the incubation. does not come in contact with the flow-through while
NOTE: The pH-indicator in Binding Buffer NTI ensures removing it from the centrifuge and the collection
optimal binding conditions with pH < 7.0. The yellow tube. (Residual ethanol from Buffer NT3 might
color facilitates to identify undissolved agarose during inhibit enzymatic reactions. Total removal of
DNA gel extraction. The optimal pH to bind even ethanol can be achieved by incubating the columns
small DNA fragments to the silica membrane of the for 2-5 min at 70 °C prior to elution.)
NucleoSpin Gel and PCR Clean-up Columns is 20) Place the NucleoSpin Gel and PCR Clean-up
approximately 5.0–6.0. The Binding Buffer NTI is Column into a new 1.5 ml microcentrifuge tube.
sufficiently buffered to maintain this pH for all 21) To elute DNA, add 15-30 μl of Buffer NE (5 mM
standard PCR reaction buffers or agarose gel buffer Tris·Cl, pH 8.5) or H2O to the center of the silica
systems. In addition, the colored binding buffer helps membrane in NucleoSpin Gel and PCR Clean-up
identify undissolved pieces of agarose during DNA gel Column and incubate at room temperature (18-25 °C)
extraction. A yellow color indicates the optimal pH < for 1 min.
6.0. If the pH increases to around 7 after adding the
22) Centrifuge the column for 1 min at 11,000 x g.
sample, the solution will turn . In case the pH is
higher th+an 7 the solution turns blue color. If a
23) Quantify the eluted DNA by using nanodrop
change in color is observed, the pH should be spectrophotometer and visualise it in a 1% agarose
adjusted by adding more Buffer NTI or by titrating the gel.
pH to < 6.0 with 4 M sodium acetate pH 5.0 or small 24) Use appropriate amount of purified DNA in
amounts of hydrochloric acid (HCl). molecular cloning experiment and store the
14) Place a NucleoSpin Gel and PCR Clean-up Column remaining DNA at -20 °C.
into a provided 2 ml collection tube and load up to
700 μl sample. RESULTS:
15) Centrifuge for 30 s at 11,000 x g. Discard flow- For PCR:
through and place the column back into the same Magnesium ion (𝑀𝑔2+) functions as a cofactor for
collection tube. (Collection tubes are reused to activity of DNA polymerases by enabling
reduce plastic waste.) incorporation of dNTPs during polymerization. In
addition, 𝑀𝑔2+ facilitates formation of the complex
16) Load remaining sample if necessary and repeat
between the primers and DNA templates by
the centrifugation step.
stabilizing negative charges on their phosphate
17) To wash silica membrane, add 700 μl Buffer NT3
backbones. The magnesium concentration often
to the NucleoSpin Gel and PCR Clean-up Column and
needs optimization to maximize PCR yield while
centrifuge for 30 s at 11,000 x g. Discard flow-through
maintaining specificity due to its binding to dNTPs,
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primers, DNA templates, and EDTA. 1.5, 2, 3, 4, 5 For Restriction Enzyme Digestion:
microliters were added to the tubes to optimize
magnesium. These samples were run on agarose gel.
The agarose gel result is given below.
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PCR product, the plasmid DNA and the marker, more than big fragment because of small fragment
formed bands at common points. spreads light more than big fragment for 4 μl of
magnesium chloride. This may be due to non-specific
FOR AGAROSE GEL ELECTROPHORESIS AND DNA primer-template compatibility, or excessive
ISOLATION FROM AGAROSE GEL: magnesium may inhibit DNA polymerase or the
sample can be added wrongly to agarose gel for 5 μl
DNA isolation is a critical step in molecular biology. It is of magnesium chloride.
necessary to obtain a specific DNA fragment from the
extracted DNA in molecular biology techniques. After For Restriction Enzyme Digestion:
isolating plasmids it may contain some chromosomal DNA
contamination it will interrupt the further processing of Restriction enzyme, also called restriction
cloning. So it is better to recover the plasmid DNA by endonuclease, a protein produced by bacteria that
eluting it from agarose gels. The first step in extracting cleaves DNA at specific sites along the molecule. In
DNA is identifying the DNA band which is to extract, by
the bacterial cell, restriction enzymes cleave foreign
illuminating under UV light. The desired band is then
DNA, thus eliminating infecting organisms.
carefully cut by a Scalpel blade. The results of this
In this experiment, according to the result of the
experiment are not yet complete.
agarose gel, the PCR products banded at the same
DISCUSSION: point, but the PCR product in the second column is
more fuzzy because the sample can not be loaded in
For PCR: sufficient amount in the second hole of the agarose
gel.
The polymerase chain reaction (PCR) is a relatively
FOR AGAROSE GEL ELECTROPHORESIS AND DNA
simple technique that amplifies a DNA template to
ISOLATION FROM AGAROSE GEL:
produce specific DNA fragments in vitro. Traditional
The results of this experiment are not yet complete…
methods of cloning a DNA sequence into a vector and
replicating it in a living cell often require days or
weeks of work, but amplification of DNA sequences
by PCR requires only hours. In this experiment, the
amount of magnesium can be insufficient or the
REFERENCES:
sample can be added wrongly to agarose gel for 1,5 μl
of magnesium chloride. The amount of magnesium 1)http://vlab.amrita.edu/?sub=3&brch=77&si
can be sufficient and the fragments can be occured m=1365&cnt=1
two different sizes such as big fragment and small
fragment but the concentration of small fragment is 2) www.yourgenome.org
more than big fragment because of small fragment
3) www.worldwide.promega.com
spreads light more than big fragment for 2 μl of
magnesium chloride. This may be due to non-specific 4) www.blog.quartzy.com
primer-template compatibility, or excessive
magnesium may inhibit DNA polymerase or the 5) www.sciencing.com
sample can be added wrongly to agarose gel for 3 μl
of magnesium chloride. The amount of magnesium 6) www.onlinelibrary.wiley.com
can be sufficient and the fragments can be occured
two different sizes such as big fragment and small
fragment but the concentration of small fragment is
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