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Gebze Technical University

Molecular Genetics Laboratory Report - 2



An alternative to cloning, called the polymerase chain reaction(PCR), can be used to directly amplify rare
specific DNA sequences in a complex mixture when the ends of the sequence are known. This method of
amplifying rare sequences from a mixture has numerous applications in basic research, human genetics
testing, and forensics.
The ability of restriction enzymes to reproducibly cut DNA at specific sequences has led to the
widespread use of these tools in many molecular genetics techniques. Restriction enzymes can be used to
map DNA fragments or genomes.
In this experiment, the effect of the amount of 𝑴𝒈𝑪𝒍𝟐 substance on PCR was researched. In addition,
pUC19 and the PCR product were cut with HindIII and EcoRI restriction enzymes.The aim of this
experiment is to observe of effect of 𝑴𝒈𝑪𝒍𝟐 on the PCR products, at the same time cut the plasmid
vector and the PCR product with restriction enzymes. Restriction digestion products are added to the
agarose gel and cutting products are observed with UV light. Then, the desired DNA fragments are
excised from the agarose gel with the help of UV light.

INTRODUCTION: of a phosphate group, a sugar (deoxyribose) and one

of four nitrogen containing bases (adenine, thymine,
guanine or cytosine). Each strand runs in the opposite
The ability to use a tiny piece of DNA and copy it
direction to the other, with only certain bases lying
millions of times via PCR has transformed molecular
opposite each other. This means that the two strands
biology. Testing for genetic backgrounds and genetic
are complementary to each other in the order of their
defects requires only a small sample, yet it yields vast
bases. The two strands of DNA are held together by
amounts of crucial information that aid medicine and hydrogen bonding between nucleotide base pairs.
ancestry research. PCR was also used to detect HIV in Hydrogen bonds are much weaker than the covalent
human cells, opening the field of epidemiology to the bonds which link individual nucleotides within a
benefits of rapid DNA amplification. Forensic strand and may be disrupted by heating the DNA.
scientists regularly use PCR, isolating DNA evidence Therefore, we can separate t he two strands
from strands of hair or small samples of blood, and of DNA without breaking the DNA strands down by
thereby aiding in fighting crime. Even fossils can yield heating to around 95°C – this process is
fragments of DNA that can be replicated many times, called denaturation. Primers are short sequences of
providing information about evolution. The power of complementary DNA which bind to
heat-stable Taq polymerase has led to a vast and certain nucleotide sequences along the DNA strand.
priceless advancement in science. The structure They tend to bind onto the single DNA strands at
of DNA consists of two strands of chains of molecules higher temperatures than the entire complementary
called nucleotides. Each of these nucleotides consists strand. This means that if the temperature is cooled
from 95°C to around 50-60°C, the primers will bind to
the single strands before the complementary whole Template DNA:
strands do. This process is called annealing. The A PCR template for replication can be of any DNA
production of a new complementary strand source, such as genomic DNA (gDNA) ,
of DNA using a single strand is performed by a class complementary DNA (cDNA), and plasmid DNA.
of enzymes called polymerases.These enzymes start Nevertheless, the composition or complexity of the
off by binding to the primers and then extend DNA contributes to optimal input amounts for PCR
the primers by adding new nucleotides to the 3’ end,
amplification. 0.1–1 ng of plasmid DNA is sufficient,
using the single stranded DNA as a template. Most
while 5–50 ng of gDNA may be required as a starting
polymerases function best at the temperatures that
amount in a 50 µL PCR. Optimal template amounts
their cells operate at. However at this temperature,
can also vary based on the type of DNA polymerase
most of the entire complementary strands will also
reattach and interfere with the function of the used; a DNA polymerase have higher sensitivity due
polymerase. There are some organisms which operate to affinity to the template would require less input
at much higher temperatures – Thermus aquaticus is a DNA. Optimization of DNA input is important because
bacterium which lives in boiling hot springs. The higher amounts increase the risk of nonspecific
polymerases which it uses operate best at around amplification whereas lower amounts reduce yields.
72°C. Therefore these enzymes may be used to ensure
that the strands are kept separate during the extension DNA polymerase:
process. DNA polymerases are critical players in replicating the
target DNA. Taq DNA polymerase is arguably the
Designing the Primers: best-known enzyme used for PCR—its discovery
Firstly, we need to know the sequence of the section revolutionized PCR. Taq DNA polymerase has
of DNA we are wanting to amplify, particularly the relatively high thermostability, with a half-life of
“beginning” (5’) and “ending” (3’) of the sequence. approximately 40 min at 95°C. It incorporates
The primers need to be designed so that they are nucleotides at a rate of about 60 bases per second at
complementary to a unique sequence of nucleotides 70°C and can amplify lengths of about 5 kb, so it is
“upstream” and “downstream of the sequence of suitable for standard PCR without special
interest. They cannot match a sequence within the requirements. For more specialized applications such
area of interest, and they should also not have as PCR cloning, long amplification, and GC-rich PCR,
complementary regions within themselves or they DNA polymerases with higher performance are
will fold over and bind to themselves, forming a preferred. These enzymes are capable of generating
“hairpin”. Lastly, the forward and reverse primers lower-error PCR products from long templates in a
should not be complementary, or they will anneal to shorter time with better yields and higher resistance
each other and form a “primer dimer”. Primers that to inhibitors.
have to 15-20 nucleotides are more useful. Deoxynucleoside triphosphates (dNTPs):
dNTPs consist of four basic nucleotides—dATP, dCTP,
The success of PCR depends on a number of factors, dGTP, and dTTP—as building blocks of new DNA
with its reaction components playing critical roles in strands. These four nucleotides are typically added to
amplification. These componenets are template DNA, the PCR reaction in equimolar amounts for optimal
DNA polymerase, primers, Deoxynucleoside base incorporation. However, in certain situations
triphosphates (dNTPs), required cofactor ( Mg ), such as random mutagenesis by PCR, unbalanced
buffer. dNTP concentrations are intentionally supplied to
promote a higher degree of misincorporation by a
non-proofreading DNA polymerase.
𝟐+ There are three different types of restriction
Required cofactor ( 𝑴𝒈 ):
Magnesium ion (𝑀𝑔2+) functions as a cofactor for enzymes. Type I cuts DNA at random locations as far
activity of DNA polymerases by enabling as 1000 or more base-pairs from the recognition site.
incorporation of dNTPs during polymerization. The Type III cuts at approximately 25 base-pairs from the
magnesium ions at the enzyme’s active site catalyze site. Types I and III require ATP and may be large
phosphodiester bond formation between the 3′-OH enzymes with multiple sub-units. Type II enzymes,
of a primer and the phosphate group of a dNTP. In which are predominantly used in biotechnology, cut
addition, 𝑀𝑔2+ facilitates formation of the complex DNA within the recognized sequence without the
between the primers and DNA templates by need for ATP and are smaller and simpler. Type II
stabilizing negative charges on their phosphate restriction enzymes are named according to the
backbones. The magnesium concentration often bacterial species from which they are isolated. For
needs optimization to maximize PCR yield while example, the enzyme EcoRI was isolated from E. coli.
maintaining specificity due to its binding to dNTPs, Type II restriction enzymes can generate two
primers, DNA templates, and EDTA. Low 𝑀𝑔2+ different types of cuts depending on whether they
concentrations result in little or no PCR product, due cut both strands at the center of the recognition
to the polymerase’s reduced activity. On the other sequence or each strand closer to one end of the
hand, high 𝑀𝑔2+ concentrations often produce recognition sequence. The former cut will generate
nonspecific PCR products from enhanced stability of "blunt ends" with no nucleotide overhangs. The latter
primer-template complexes, as well as increases in generates "sticky" or "blunt" ends because each
replication errors from misincorporation of dNTPs. resulting fragment of DNA has an overhang that
compliments the other fragments.
PCR is carried out in a buffer that provides a suitable
chemical environment for activity of DNA
polymerase. The buffer pH is usually between 8.0 and
9.5 and is often stabilized by Tris-HCl. Image 1: Sticky Ends


A restriction enzyme recognizes a specific nucleotide-

Image 2: Blunts Ends
pair sequence in DNA called a restriction site and
cleaves the DNA within or near that sequence. All It has been demonstrated that under extreme non-
restriction enzymes cut DNA between the carbon and standard conditions, restriction endonucleases are
the phosphate moiety of the phosphodiester bond so capable of cleaving sequences which are similar but
that fragments produced by restriction enzyme not identical to their defined recognition sequence.
digestion have phosphates and hydroxyls. Most This altered or relaxed specificity has been termed
restriction enzymes function optimally at Restriction star activity. It has been suggested that star activity
enzymes are used to produce a pool of DNA may be a general property of restriction
fragments to be cloned. Restriction enzymes are also endonucleases and that any restriction endonuclease
used to analyze the positions of restriction sites in a can be made to cleave noncanonical sites under
piece of cloned DNA or in a segment of DNA in the certain extreme conditions.
genome. Such a digest will cut each genome copy of
the same organism into the same large set of pieces.
The manner in which an specificity of enzyme is EXAMPLE: the name of EcoRI restriction enzyme is
altered depends on the enzyme and on the conditions derived as:
employed to induce the star activity. The most
E : Escherichia (genus)
common types of altered activity are single base
substitutions, truncation of the outer bases in the co : coli (species)
recognition sequence, and single-strand nicking. R : RY13 (strain)
I : first identified in the bacterium
Conditions that Contribute to Star Activity
1.High glycerol concentration In this experiment, the PCR product and the plasmid
2. High units to µg of DNA ratio vector pUC19 will be used and cut with EcoRI and
3. Low ionic strength HindIII restriction enzymes. The results will be
4. High pH monitored by agarose gel electrophoresis.
5.Presence of organic solvents
6.Substitution of 𝑀𝑔2+with other divalent cations

Inhibiting Star Activity

1. Use as few units as possible to get a complete
digestion. This avoids overdigestion and reduces the
final glycerol concentration in the reaction.
2. Make sure the reaction is free of any organic
solvents such as alcohols which might be present in
the DNA preparation.
3. Raise the ionic strength of the reaction buffer to
100-150 mM (provided the enzyme is not inhibited by
high salt).
4. Lower the pH of the reaction buffer to pH 7.0.
5. Use 𝑀𝑔2+as the divalent cation.

Image 3: Puc19 Vector

Nomenclature of restriction enzymes :
The cutting points of the Puc19 vector with EcoRI and
1. Indicates the genus and species of specific HindIII are shown in Image 3. The vector Puc19 has
organism by using abbreviation into three 2686 base pairs.
letters. Eco is for Escherichia coli; here first
letter ‘E’ is for genus whereas last two letters 3) AGAROSE GEL ELECTROPHORESIS AND DNA
‘co’ is for species. ISOLATION FROM AGAROSE GEL:
2. Indicates the strain of the relevant species by The technique of agarose gel electrophoresis has
using a letter, number or its combination. several important applications. Agarose gel
3. Indicate specific restriction modification electrophoresis is a method of separating DNA and
systems present in the same organism or RNA fragments. The agarose gel has an adjustable
strain by using a roman numeral. pore hole depending on the DNA or RNA molecule to
be used. As DNA or RNA molecules are placed on
fields with electric currents, negatively charged

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
 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
5) The tubes are waited for -80 °C for 30 min.

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
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,

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.

Image 5: Agarose Gel of Restriction Enzyme Digestion

A restriction enzyme recognizes a specific nucleotide-

pair sequence in DNA called a restriction site and
Image 4: Agarose Gel of PCR cleaves the DNA within or near that sequence. All
Low 𝑀𝑔2+ concentrations result in little or no PCR restriction enzymes cut DNA between the carbon and
product, due to the polymerase’s reduced activity. On the phosphate moiety of the phosphodiester bond so
the other hand, high 𝑀𝑔2+ concentrations often that fragments produced by restriction enzyme
produce nonspecific PCR products from enhanced digestion have phosphates and hydroxyls. In this
stability of primer-template complexes, as well as experiment, pUC19 plasmid and Pac3,5 gene were
increases in replication errors from misincorporation used for restriction cutting. The pUC19 plasmid and
of dNTPs. Any result is not seen in 1,5 μl magnesium Pac3,5 gene are cut with EcoRI and HindIII because of
chloride. The two results are seen in 2 μl magnesium these enzymes can cut same point in both of them.
chloride. 3 microlitres of magnesium chloride showed After the plasmid and PCR product DNAs are cut,
very little result. The two results are seen in 4 μl these samples added to agarose gel. These samples
magnesium chloride. 5 microlitres of magnesium were run on agarose gel. The agarose gel result is
chloride showed very little result. In addition, one of given below. According to the result of the agarose
2 microliters of magnesium chloride did not yield a gel, the PCR products banded at the same point, but
the PCR product in the second column is more fuzzy.
result. The reason of this case, the sample can be
The plasmid DNAs banded at the same point. We can
added wrongly to agarose gel.
say that, this experiment is successful because of the

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
The polymerase chain reaction (PCR) is a relatively
simple technique that amplifies a DNA template to
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
sample can be added wrongly to agarose gel for 1,5 μl
of magnesium chloride. The amount of magnesium 1)
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)
more than big fragment because of small fragment
spreads light more than big fragment for 2 μl of
magnesium chloride. This may be due to non-specific 4)
primer-template compatibility, or excessive
magnesium may inhibit DNA polymerase or the 5)
sample can be added wrongly to agarose gel for 3 μl
of magnesium chloride. The amount of magnesium 6)
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
~ 10 ~