GEBZE TECHNICAL UNIVERSITY

MOLECULAR GENETICS LABORATORY

EBRU AKHARMAN
142204026
 Molecular Cloning, Preparation of Competent Cells
And Bacterial Transformation

AIM

Molecular cloning can be accomplished by specific methods from the prokaryotic or eukaryotic
cell genome to which the gene encoding an important product or protein synthesis belongs
(usually by restriction endonuclease enzymes), which is transferred to a recipient prokaryotic
or eukaryotic cell by combining with a carrier vector DNA, provides genetic expression in the
recipient cell. Bacterial transformation is a natural process in which cells take up foreign DNA
from the environment at a low frequency. After transformation, the cells may express the
acquired genetic information, which may serve as a source of genetic diversity and potentially
provide benefits to the host (e.g., antibiotic resistance). With the molecular cloning , the process
of transformation was exploited and enhanced to introduce recombinant plasmid DNA into
bacterial strains that were made “competent,” or more permeable, for DNA uptake. The purpose
of this experiment is DNA transformation to competent cells.
INTRODUCTION
For Molecular Cloning:
DNA replication involves the copying of each strand of the double helix to give a pair of daughter strands.
Replication begins at a specific sequence, called the origin. After initiation begins at an origin sequence, all
sequences are replicated no matter what their information. This principle leads to the idea of molecular
cloning, or recombinant DNA. Cloning enables the production of a single DNA sequence in large quantities.

A recombinant DNA consists of two parts: a vector and the insert sequences. Vectors supply replication
functions the origin sequences to the recombinant DNA molecule. After it joins to a vector, any insert
sequence can be replicated. The process of joining the vector and insert DNAs is called ligation. DNA ligase
carries out ligation by using ATP energy to make the phosphodiester bond between the vector and insert. If
the vector and insert DNA fragments are produced by the action of the same restriction endonuclease, they
will join by base‐pairing. Ligation conditions. In blunt-end ligations, the association of 5’ phosphate groups and
3’ hydroxyl groups is more transient than in cohesive-end ligations. Because they lack the hydrogen bond
stabilization of cohesive ends, blunt-end ligations are more sensitive to reaction conditions, especially to the
concentrations of the reaction components. The likelihood of an insert associating with a linearized plasmid is
increased by having a high concentration of available insert blunt ends. However, the intramolecular
circularization of the plasmid, after one end of the insert has been joined, works best at lower concentrations.

T4 DNA Ligase: The most common method of covalently joining the insert and plasmid DNA ends is to use
the ATP-dependent, T4 DNA ligase. Ligation using this enzyme is usually performed between 4°C and 25°C.
Cohesive ends have an annealing temperature below room temperature because they are only a few bases
long. Low temperature ligations help stabilize DNA interactions, but at the expense of enzyme activity. The
reduced activity of T4 ligase at lower temperatures can be partially compensated for by increasing the
duration of the ligation reaction. In addition, because annealing temperature of the ends affects ligation
efficiency, cohesive-end ligations with short or single base overhangs may benefit from higher concentrations

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of T4 ligase. Follow the manufacturer’s reaction protocol for their T4 ligase and buffer. For extended
incubations, some researchers even add additional enzyme into the reaction midway through.

Image 1: Ligation

The following formula can be used to calculate the amount of insert for a specified amount of vector.

(𝒏𝒈 𝒐𝒇 𝒗𝒆𝒄𝒕𝒐𝒓 𝑿 𝒔𝒊𝒛𝒆 𝒐𝒇 𝒊𝒏𝒔𝒆𝒓𝒕 (𝒊𝒏 𝒌𝒃))

Insert mass in nanogram (ng)= 𝑿 (𝒊𝒏𝒔𝒆𝒓𝒕 ∶ 𝒗𝒆𝒄𝒕𝒐𝒓 𝒎𝒐𝒍𝒂𝒓 𝒓𝒂𝒕𝒊𝒐)
𝒔𝒊𝒛𝒆 𝒐𝒇 𝒗𝒆𝒄𝒕𝒐𝒓 (𝒊𝒏 𝒌𝒃)

For Preparation of Competent Cells:

Competent cells are ready to use bacterial cells that possess more easily altered cell walls by which foreign
DNA can be passed through easily. Most types of cells cannot take up DNA efficiently unless they have been
exposed to special chemical or electrical treatments to make them competent . The standard method for
making the bacteria permeable to DNA involves treatment with calcium ions. Brief exposure of cells to an
electric field also allows the bacteria to take up DNA and this process is called as electroporation .

However, some types of bacteria are naturally transformable, which means they can take up DNA from their
environment without requiring special treatment. The exact mechanisms involved in artificial competence are
not yet known well. ln 𝑀𝑔𝐶𝑙2 method, the competency can be obtained by creating pores in bacterial cells
by suspending them in a solution containing high concentration of calcium. DNA can then forced in to the Host
cell by heat shock treatment at 42𝑜 𝐶 for the process of transformation.

Natural Competence: Bacteria are able to take up DNA from their environment by three ways; conjugation,
transformation, and transduction. In transformation the DNA is directly entered to the cell. Uptake of
transforming DNA requires the recipient cells to be in a specialized physiological state called competent state.
It is highly regulated in bacteria, and the factors involved in competence vary among genera. The competence
proteins produced have some homology but differ in the Gram negative and the Gram positive bacteria.
Once the DNA has been brought into the cell's cytoplasm, it may be degraded by the nuclease enzymes, or, if
it is very similar to the cells own DNA, the DNA repairing enzymes may recombine it with the chromosome.

Artificial Competence and Transformation: Artificial competence is not encoded in the cell's genes. Instead it
is a laboratory procedure by which cells are made permeable to DNA, with conditions that do not normally

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occur in nature. This procedure is comparatively easy and simple, and can be used in the genetic engineering
of bacteria but in general transformation efficiency is low. There are two main methods for the preparation of
competent cells. They are Calcium chloride method and Electroporation.

Image 2: Prepairing Competence Cell

Rapidly growing cells are made competent more easily than cells in other Growth stages. So it is necessary to
brought cells into log phase before the procedure is begun. The cells in rapid growth (log phase) are living,
healthy, and actively metabolizing.

TSS (Transformation and Storage Solution for Chemical Transformation):

We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses
a transformation and storage solution [TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5%
(vol/vol) dimethyl sulfoxide, and 50 mM 𝑀𝑔2+at pH 6.5]. Cells are mixed with an equal volume of ice-cold 2 x
TSS and are immediately ready for use. Genetic transformation is equally simple: plasmid DNA is added and
the cells are incubated for 5-60 min at 4 degrees C. A heat pulse is not necessary and the incubation time at 4
degrees C is not crucial, so there are no critical timing steps in the transformation procedure. Transformed
bacteria are grown and selected by standard methods.

Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current
methods. Although cells taken early in the growth cycle (𝑂𝐷600 0.3-0.4) yield the highest transformation
efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the
growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7)
transformants per micrograms of DNA). For long term storage of competent cells, bacteria can be frozen in
TSS without addition of other components. Our procedure represents a simple and convenient method for the
preparation, transformation, and storage of competent bacterial cells.

Polyethylene Glycol (PEG) is a polyether compound having many functions. In this case, it helps in shielding
the negative charges present on the DNA and host cell membrane. This results in lowering of repulsion. In
addition, PEG, being a larger molecule, coordinates with the water molecules present in the bacterial
suspension. This results in the increased concentration and bioavailability of the plasmid DNA to pass through
the membrane of a bacterial competent cell. In other words, a highly effective plasmid concentration results
in a more effective DNA transformation into bacteria.

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Dimethyl Sulfoxide (DMSO) is an organosulfur compound and is a polar aprotic solvent. It can be dissolved
both in polar and nonpolar compounds and is miscible with a wide range of organic solvents as well as water.
DMSO brings together the reagents that have been added during the reaction. It also acts as a preserving
agent as the prepared competent cells need to be stored at −80°C for a longer period without losing their
viability.

𝑴𝒈𝑪𝒍𝟐 acts in the same way as does 𝐶𝑎𝐶𝑙2 . It induces the ability of the cells to take up DNA by altering
permeability of the membranes. The negatively charged incoming DNA is repelled by the negatively charged
macromolecules present on the bacterium’s outer surface which is neutralized by the addition of 𝑀𝑔𝐶𝑙2 to
neutralize the unfavourable interactions.

For Bacterial Transformation:

Foreign DNA can be placed in cells by several methods. If the foreign DNA is introduced into the cell in a form
acceptable to the host, genes on that DNA can be expressed and the DNA can be propagated by the cells. In
many cases this is done by attaching the foreign DNA to a piece of DNA that is capable of replicating within
the host. For bacteria and some eukaryote species, plasmids or phages represent suitable vectors.

Plasmids can carry only relatively small segments of DNA (<15 kb) but phage or cosmid vectors can carry up to
50 kb. Yeast artificial chromosomes (YAC) can be used to propagate very large foreign DNA fragments (>200
kb) in yeast. Phage or virus particles are available to transfer DNA into almost any type of cell. In other cases,
foreign DNA can be introduced without any attached vector, and can sometimes integrate itself into the host
chromosome where it is replicated as part of the host genome.

The overall process of changing the phenotype of a bacterium by introducing a plasmid into it is called
transformation. Bacteria may be transformed with plasmids by several techniques. The simplest is merely
incubating the plasmid with bacteria whose cell wall has been weakened.

 Positive Control : transform competent cells with plasmid DNA (not digested); provides measure
of the efficiency of transformation and serves as a standard for comparison with other
transformations

 Negative Controls
1. No DNA -- transform competent cells, without added DNA, and plate on selection media; if
high background, may indicate a problem with the antibiotic in the agar plates
2. Digestion efficiency -- transform competent cells with digested plasmid DNA / NO ligase
treatment; if high background, may indicate that the digestion did not go to completion
3. Vector recircularization -- transform competent cells with digested vector DNA that was
treated with alkaline phosphatase before adding ligase (NO insert is present); if high
background, may indicate that the dephosphorylation reaction was incomplete

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 Image 3: Transformation

MATERIAL AND METHOD

For Molecular Cloning:

 Cloning vector (digested with appropriate REs)

 Insert DNA (digested with the same REs as vector)
 T4 DNA Ligase (1 U/μL)
 T4 DNA Ligase Buffer (10 X)
 ATP (10 mM)

Components RXN1(insert+ vector) RXN2(vector)

Vector(0,075 μg/ μL) 1 μL 1 μL

Insert(0,1 μg/ μL) 2,9 μL -

T4 DNA Ligase Buffer 3 μL 3 μL

(from 10X to 1X)
ATP(10 mM) 1,5 μL 1 μL

𝒅𝑯𝟐 𝑶 21,1 μL 24 μL
(the volume is completed to 30 μL)
T4 DNA Ligase( 1U/ μL ) 0,5 μL 0,5 μL

Total Volume 30 μL 30 μL

 Combine the materials (except T4 DNA Ligase) given in the table below in a 1.5 ml eppendorf tube (on
ice). Prepare a control reaction with no insert and add 5,8 μl sterile water instead of insert.
 Gently mix the tubes and after a short spin incubate them at 37 °C for 10 minutes.
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  Put the tubes in ice-water bath (0 °C ) and wait for 10 minutes.
 Add 1 μl T4 DNA Ligase (1 U/μl) to each reaction tube and mix gently. After a short spin put the tubes
in ice-water bath again and wait for 30 minutes.
 Place the tubes in water bath adjusted to 16 °C and incubate them overnight
(Ligation of cohesive ends is usually carried out at 12-16 °C to maintain a good balance between
annealing of the ends and activity of the enzyme. Higher temperatures make it difficult for the ends to
anneal, whereas lower temperatures diminish ligase activity. Blunt-end ligations are typically performed at
room temperature since annealing is not a factor. T4 DNA Ligase is not stable above 30 °C.)
 Ligation reaction is stopped by incubating the tubes at 70 °C for 10 minutes.
 After a short spin, ligation mixtures should be used immediately for transformation or can be stored at
-20 °C for a few days.

For Preparation of Competent Cells:

Transformation and Storage Solution for Chemical Transformation (TSS: This solution can be stored at 4 oC
for two weeks after autoclaved.)

 LB (Tryptone 1 %, Yeast Extract 0.5 %, NaCl 1 %)

 % 10 PEG 8000 (W/V)
 % 5 DMSO (V/V)
 50 mM MgCl2, pH 6.5

 Streak the cells from -20𝑜 𝐶. stock on a LB plate. Incubate the plate at 37𝑜 𝐶 over night.
 Pick a single, well-isolated colony and inoculate it into 5 ml of LB broth. Incubate at 37𝑜 𝐶 overnight
with shaking at 200 rpm.
 Transfer 1 ml of the saturated overnight culture to a sterile 500 ml flask containing 100 ml of LB
medium (do not add antibiotic at this step). Incubate the cells at 37𝑜 𝐶 with shaking at 200 rpm, until
OD600 reach 0.5; this usually takes 2.0-2.5 hr. Check the OD frequently when it gets beyond 0.2 to
avoid overgrowth.
(This procedure requires that cells be growing rapidly (early- or mid-log phase). Accordingly, it is very
important that the growing cells have sufficient air. Overgrowth of culture decreases the efficiency of
transformation.)
 When the culture reaches an OD600 of 0.5, chill the flask on ice for 20 min and then collect the cells by
centrifugation at 7000 rpm for 10 min at 4𝑜 𝐶. (Cells should be kept cold for all subsequent steps.)
The OD value represents the amount of light that is absorbed by your sample. But that value is affected by
the intensity of the light beam in the spec, and the spec design. This means that similar samples will give
completely different OD values in different specs due to the specs having different bulbs, or even in the
same spec over time, as the beam intensity reduces with the age of the bulb.
 Pour off supernatant and resuspend each pellet in 10 ml of ice-cold TSS solution. Now the competent
cells are ready to be transformed. (Resuspension should be performed very gently and all cells kept
on ice.)
 If they are not immediately used, cells can be stored at 4𝑜 𝐶. for maximum of 6 hr without significant loss
of competency. The same competent cells can also be stored at -70𝑜 𝐶. for long-term storage.
Transformation frequency of frozen cells is 30 % of that of the fresh cells, when used within two months.

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 Transformation efficiency = The number of transformant colonies counted on the plate / The amount of DNA
transformed (μg)

The amount of DNA transformed (μg) = The amount of DNA transformed (ng) x 10−3

For Bacterial Transformation:

 Competent cells (E. coli JM 109)
 Ligation materials (from the molecular cloning experiment)
 RXN 1 (insert + vector) (pUC 19 plasmid vector + insert DNA; both of which were digested
with the same restriction enzymes)
 RXN 2 (vector) (pUC 19 plasmid vector which was digested with the same restriction enzymes
as insert DNA)
 pUC 19 plasmid DNA (nondigested) (will be used as positive control)
 Firstly, take the competent cells from -70 °C freezer and thaw on ice.
 Pipette 5 μl of each ligation material into 1,5 ml epp tubes separately and place on ice. (Ligation
material 2 (RXN 2) is the negative control of ligation reaction and will be used to specify the number
of background colonies.)
 Put 1; 10; 25; 50 and 100 ng of pUC 19 plasmid DNA (nondigested) into different 1,5 ml epp tubes and
place on ice. (The result of this transformation will be used to evaluate the competency of the
bacterial cells.)
 Add 100 μl competent cells, thawed on ice, into each epp tube containing DNA. Prepare also a control
tube containing competent cells only (The control tube should be prepared firstly).
When handling frozen competent cells, it is important to keep it cool! Frozen cells are very sensitive to
being warm and will not work as well if not kept on ice as much as possible. Competent cells should be used
immediately after thawing. Remaining cells should be discarded rather than refrozen.
 Gently swirl tubes to mix, then place on ice for 30 mins.
 Heat shock cells by placing the tubes into a 42 °C water bath for 2 min without shaking.
The heat shock step is necessary for the uptake of DNA. At temperatures above 42 °C, the bacteria's
ability to uptake DNA becomes reduced, and at extreme temperatures the bacteria will die.
 Put the tubes on ice for 2 min immediately.
 Add 1 ml LB medium (RT) to each tube.
 Place the tubes on a shaker at 200 rpm for 1 hr at 37 °C.
 Spin the epp tubes for 30 seconds and decant the supernatant.
 Resuspend the pellet in the remaining LB medium (around 100 μl) and spread onto LB/ampicillin
containing plates.
 Incubate the plates overnight (12 to 16 hr) at 37 °C.
 Next day morning, take the petri plates, count the colonies and evaluate the result. The expected
transformation frequency should be in the range of 𝟏𝟎𝟔 – 𝟏𝟎𝟕 transformants / μg DNA.
 Use 1-100 ng of pUC 19 to transform 100 μl of competent cells according to the standard protocol.
Plate all the transformation culture (after pelleting and then resuspending in an appropriate volume
of medium) on LB/ampicillin plates and incubate at 37°C overnight. (100 μg of ampicilin is added for 1
ml of LB. Thus, 3 ml of ampicilin is added for 300 ml of LB.) LB agar should be cold when ampicillin is
added. Otherwise, the antibiotic will fail to function and the experiment will disrupt.

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 Steps Ligation Ligation Control Negative Control Compotent Cell

1; 10; 25; 50 and

RXN1 RXN2 E. coli JM 109 100 ng of pUC 19
(vector+insert) (only vector) plasmid DNA

5 μl of RXN1 and 100 5 μl of RXN2 and 5 μl of E. coli JM 1 μl of pUC 19

First Step μl of LB Broth 100 μl of LB Broth 109 and 100 μl of plasmid DNA and
LB Broth 100 μl of LB Broth
100 μl of first step is 100 μl of first step 100 μl of first step 100 μl of first step is
Second Step added on LB agar + is added on LB is added on LB agar added on LB agar +
ampicilin agar + ampicilin + ampicilin ampicilin

RESULTS

Image 4: Ligation Control 1 Image 5: Ligation Control 2

The ligation control is RXN2. There are competent
bacterias containing only the vector DNA. These
cells do not contain the pUC 19 sequence and thus
are not antibiotic resistant. However, these bacteria
have formed colonies in LB agar which has 100 μg /
ml of antibiotic.
The ligation control is RXN2. There are competent
bacterias containing only the vector DNA. These
cells do not contain the pUC 19 sequence and thus
are not antibiotic resistant. These bacterias are not
survive because of they have not resistance gene in
this petri. This is the expected result.

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 Image 6: Negative Control Image 7: Ligation 1
The E. coli JM109 bacterial colonies could be formed The ligation1 is RXN1. There are competent bacterias
in LB agar which has 100 μg / ml of antibiotic. containing the vector and insert DNA. These cells contain
the pUC 19 sequence and thus are antibiotic resistant.
Thus, these bacteria have formed colonies of LB agar which
has 100 μg / ml of antibiotic. This indicates that the ligation
has been successful.

Image 8: Ligation 2 Image 9: 1 ng of pUC 19 plasmid DNA

The ligation2 is RXN1. There are competent bacterias
containing the vector and insert DNA. These cells contain 43 colonies are observed with 1 ng of the pUC 19 plasmid
the 19 sequence and thus are antibiotic resistant. Thus, DNA. 1 ng of the pUC 19 plasmid DNA has 43.000 μg of
these bacteria have formed colonies of LB agar which has transformation efficiency.
100 μg / ml of antibiotic. This indicates that the ligation has
been successful.
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 Image 10: 10 ng of pUC 19 plasmid DNA Image 11: 25 ng of pUC 19 plasmid DNA

180 colonies are observed with 10 ng of the pUC 19 880 colonies are observed with 25 ng of the pUC 19
plasmid DNA. 10 ng of the pUC 19 plasmid DNA has 18.000 plasmid DNA. 25 ng of the pUC 19 plasmid DNA has
μg of transformation efficiency. 35.200 μg of transformation efficiency.

Image 12: 50 ng of pUC 19 plasmid DNA Image 13: 100 ng of pUC 19 plasmid DNA

892 colonies are observed with 50 ng of the pUC 19 1300 colonies are observed with 100 ng of the pUC 19
plasmid DNA. 50 ng of the pUC 19 plasmid DNA has 17.840 plasmid DNA. 100 ng of the pUC 19 plasmid DNA has
μg of transformation efficiency. 13.000 μg of transformation efficiency.

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Calculations:

Image 14: Calculating of Bacterial Colonies

Transformation efficiency = The number of transformant colonies counted on the plate / The amount of DNA
transformed (μg)

The amount of DNA transformed (μg) = The amount of DNA transformed (ng) x 10−3

For 1 ng of pUC 19 plasmid DNA:

The amount of DNA transformed (μg) = 1 ng is 10−3 μg

The number of transformant colonies counted on the plate = 43

𝟒𝟑
= 43 x 10+3 =43.000 μg
𝟏𝟎−𝟑

For 10 ng of pUC 19 plasmid DNA:

The amount of DNA transformed (μg) = 10 ng is 10−2 μg

The number of transformant colonies counted on the plate = 180

𝟏𝟖𝟎
= 180 x 10+2 =18.000 μg
𝟏𝟎−𝟐

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For 25 ng of pUC 19 plasmid DNA:

The amount of DNA transformed (μg) = 25 ng is 25𝑥10−3 μg

The number of transformant colonies counted on the plate = 880

𝟖𝟖𝟎 𝟖𝟖𝟎 𝐱 10+3

= =35.200 μg
𝟐𝟓𝐱𝟏𝟎 −𝟑
𝟐𝟓

For 50 ng of pUC 19 plasmid DNA:

The amount of DNA transformed (μg) = 50 ng is 50𝑥10−3 μg

The number of transformant colonies counted on the plate = 892

𝟖𝟗𝟐 𝟖𝟗𝟐 𝐱 10+3

= =17.840 μg
𝟓𝟎𝐱𝟏𝟎 −𝟑
𝟓𝟎

For 100 ng of pUC 19 plasmid DNA:

The amount of DNA transformed (μg) = 100 ng is 10−1 μg

The number of transformant colonies counted on the plate = 1300

𝟏𝟑𝟎𝟎
= 1300 x 10+1 =13.000 μg
𝟏𝟎−𝟏

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 DISCUSSION

A process of producing multiple and identical copies of a particular section of DNA is called as DNA cloning.
During this technique, the selected DNA fragment is inserted into a plasmid (the circular piece of DNA) using
enzymes. The enzymes used in this process are called as restriction enzymes and DNA ligase enzymes. These
restriction enzymes, naturally found in bacteria are used to cut the DNA fragments at specific sequences and
DNA ligase enzymes are used to paste or rejoin the DNA fragments with complementary ends. The produced
molecule of recombinant DNA is introduced into a selected and grown up bacteria. These bacteria reproduce
by producing an exact copy of the plasmid and transferring it to their progeny, making copies of the DNA it
contains. The DNA molecules produced through the cloning techniques are used for many purposes which
includes:

 There is need of more DNA copies for studying about the different gene, its functions, the cause of
different diseases and for other research and experiments.
 Cloning livestock, and for reviving endangered or extinct species.
 Gene therapy used to treat some genetic disorders and replacement of a particular gene.
 In pharmaceuticals industries for the production of recombinant protein to produce human proteins
with biomedical applications.

The plasmid prepared in this experiment was transferred to competent cells. However, various processes have
been carried out to check the efficiency of the transformation process. Ligation control, negative and positive
control are some of these. There are competent bacterias containing only the vector DNA in ligation control.
1. These cells do not contain the pUC 19 sequence and thus are not antibiotic resistant. However, these
bacteria have formed colonies of LB agar which has 100 μg / ml of antibiotic. The cause of this situation may
be contamination. The glass bag was not sterilized as much as it was during the process, so a colony may have
formed. These bacterias are not survive because of they have not resistance gene in this petri in ligation
control 2. This is the expected result. The E. coli JM109 bacterial colonies could be formed in LB agar which
has 100 μg / ml of antibiotic in negative control. The E. coli bacterium has been made resistant to antibiotics
and the antibiotic used may have been disrupted. There are competent bacterias containing the vector and
insert DNA in ligation 1 and 2. These cells contain the pUC 19 sequence and thus are antibiotic resistant. Thus,
these bacteria have formed colonies of LB agar which has 100 μg / ml of antibiotic. This indicates that the
ligation has been successful. The ratio of the number of transformant colonies counted on the plate and the
amount of DNA transformed gives transformation effiency. According to this process, transformation effiency
of 1 ng plasmid DNA is 43,000 μg, transformation effiency of 10 ng plasmid DNA is 18,000 μg , transformation
effiency of 25 ng plasmid DNA is 35,200 μg, transformation effiency of 50 ng plasmid DNA is 17,840 μg,
transformation effiency of 100 ng plasmid DNA is 13,000 μg. The 1 ng pUC 19 plasmid DNA has the highest
transformation effiency.

RESOURCES

www.byjus.com

www.medicine.jrank.org

www.unitconversion.org
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