Experiment #1: E.

Coli DNA Isolation and UV

Quantitation

1
Introduction:
The purpose of this experiment was to break down HB101 E. Coli cells and separate the
cell compartments down in order to isolate DNA from all other cellular components. Once
isolated, the DNA was extracted and then tested to see how much of the DNA was pure and
present in a single bacterial cell. The four steps to isolating DNA is cell growth, cell harvest and
lysis, DNA purification, and DNA concentration. The E. Coli cells were grown overnight using a
BHI media. The BHI media is brain and heart infused. The purpose of this was to determine the
number of bacteria in culture. Some of the cells may have surpassed the growth stage and have
already gone into the death phase. After the cells were grown, they were taken out in order to do
a serial dilution. The diluted samples were then grown on a plate and began to grow bacterial
colonies. The rest of the cells that are harvested are centrifuged. The purpose of this is to
physically separate the cells from the BHI media. After centrifugation, the next step is cell lysis.
This physically breaks open the cell and allows the inner contents of the cell to be exposed. In
order to break down the cell wall and membrane, EDTA and lysozyme was used. Once the cell is
lysed, the cellular components is separated from other cells parts such as RNA and proteins by
using RNAase, SDS, and sodium percolate. These products allow bonds to break, proteins to
denature and proteins to dissociate from the isolated DNA. Phase separation is then used to
create a distinction between DNA, proteins, and lipids. Centrifugation and chloroform alcohol
also assists in the distinct layers seen in phase separation. One can see DNA located at the top of
the tube in an aqueous phase. This aqueous layer is separated by using a pipette and then
transferred into a tube. The DNA can then be precipitated out using ethanol. By mixing well, one
can use a glass rod to transfer the string of DNA into a culture tube containing water. Dilutions
of the DNA are then made and separated into three samples. These samples are then places in a
spectrophotometer in order to obtain absorbance values at both wavelength values, 260nm and
280nm. The data can then be applied to Beer Lambert Law in order to quantify how much of
pure DNA is isolated.
Genomics is the study of the genome or more specifically, DNA, of an organism.
Genomics has evolved vastly to a point where now the scientific advances can allow researchers
to decipher the DNA sequence of the whole genome of an organism. By doing so, researchers
can compare the genome of different species and analyze what genes contribute phenotypic
features. By isolating DNA, researchers can separate a specific region genome and produce
unlimited number of copies of it. Researchers can use their copies to study and manipulate DNA,
RNA, and protein and test the effects in both vivo and vitro. Some of the most important
techniques used is DNA cloning. By isolating a single DNA molecule, researchers can utilize
cloning vectors in order to copy and generate over millions of identical molecules. By using
restriction nucleases, scientists can cleave specific sites of DNA in order to isolate and
manipulate individual genes. This has led to the discovery of new classes of genes and proteins.
An isolated altered gene can be transferred back into the germ line of the organism and tests the
effects it may have on an organism’s genome. (Smith/ Tolstoi)

Methods:
All of the solutions used in this experiment plays a crucial role in the success of purifying
and isolating DNA from the bacterial cell. The very first solution seen in the protocol is saline-
EDTA. The purpose of EDTA is to bind onto cations and structurally form cages around cations.
Ultimately, EDTA chelates metal cations and makes the outer membrane of E. Coli unstable.
EDTA also serves to prevent DNAse 1 by taking and binding to magnesium and calcium.
DNAse 1 is activated when bound to both Mg2+ and Ca2+ and once activated degrades DNA.
2
The second solution used is lysozyme. Lysozyme is an enzyme that attacks the peptidoglycan
layer. The peptidoglycan layer consists of many sugar units bound by a Nag—Nam bond. The
lysozyme cleaves the glycosidic bond between Nag—Nam and assists to the lysis of the E. Coli
bacterial cell. The third solution used is RNAseA. RNAseA is used here to degrade single
stranded RNA nucleic acids. It specifically cleaves on the 3’ side of phosphodiester bonds. This
also can assist in assuring that we are isolating DNA alone and not RNA. The forth solution used
is 20% SDS. SDS is an anionic detergent that works by using its long hydrophobic tails and
charged heads to insert into the membrane and solubilizing the lipid bilayer while disrupting the
cell membrane and denaturing enzymes. The fifth solution used in the protocol is sodium
perchlorate. At this point of the experiment, the DNA collected is not pure. It contains lipids,
polysaccharides, proteins, and DNA. In order to purify further, proteins need to be stripped
away. The sodium perchlorate has such a high salt concentration that it assists in inhibiting
interactions between DNA and other associated proteins. It is also found to be helpful in later
steps while precipitating DNA in an alcohol solution. The sixth solution used is chloroform-
isoamyl alcohol. Chloroform removes proteins and lipids from the aqueous phase in order to
assure that the DNA content is in fact pure. The isoamly alcohol helps in the phase separation.
The seventh significant solution used is 100% ethanol. Ethanol allows DNA to precipitate out of
solution. Nucleic acids will precipitate in alcohol in the presence of salts. This is where the salt,
sodium perchlorate, also serves another important role. This concludes the important solutions
used in order to isolate and purify the DNA from a bacterial cell.

Results:
In order to quantify the amount of genomic DNA isolated from E. Coli cells, the precipitated
DNA is collected on a glass rod and places into a tube with 4ml of water. This creates a
concentrated sample. In order to suspend DNA out of this concentrated sample, the tube was
vortexed. This created an aqueous solution that is used to make three samples each with different
dilution factors. Table 1 shows the three samples that were created.

Table 1:
Dilution Factor of Samples

DNA sample Water Dilution Factor

Sample A 100 ul 900 ul 10
Sample B 20 ul 980 ul 50
Sample C 10 ul 990 ul 100

The dilution factor can be calculated by using the final volume and the volume of DNA solution
that was transferred. The calculations below show the work for all three of the samples.

Equation 1:
Final volume
Dilution Factor = Aliquot volume

100 𝑢𝑙 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝐷𝑁𝐴 + 900 𝑢𝑙 𝑑𝐻2 𝑂 1000

Sample A: Dilution Factor = = = 10(101 )𝐷. 𝐹.
100 𝑢𝑙 𝑜𝑓 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝐷𝑁𝐴 100
20 𝑢𝑙 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝐷𝑁𝐴 + 980 𝑢𝑙 𝑑𝐻2 𝑂 1000
Sample B: Dilution Factor = = = 50 𝐷. 𝐹.
20 𝑢𝑙 𝑜𝑓 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝐷𝑁𝐴 20

3
 10 𝑢𝑙 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝐷𝑁𝐴 + 990 𝑢𝑙 𝑑𝐻2 𝑂 1000
Sample C: Dilution Factor = = = 100(102 )𝐷. 𝐹.
10 𝑢𝑙 𝑜𝑓 𝑜𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝐷𝑁𝐴 10

These three samples were then put into the spectrophotometer and absorbance values were
recorded at both wavelength values, 260nm and 280nm. The reason for using both of these
wavelength values is due to the fact that DNA hits its maximum peak at 260nm while proteins do
at 280nm. The ratio of these absorbances determine if there was any protein contamination.
Typically, a ratio between 1.8 and 2.0 indicates little protein contamination. Table 2 provides the
absorbance values and ratio values for all three samples.

Table 2:
Absorbance Value of DNA

260 nM 280 nM 𝟐𝟔𝟎

𝑹𝒂𝒕𝒊𝒐
𝟐𝟖𝟎
Sample A 0.150 A 0.078 A 1.923
Sample B 0.032 A 0.017 A 1.882
Sample C 0.018 A 0.009 A 2

260
All three of the samples provided good readings that were in between 1.8 and 2.0 in the 280 ratio.
However, he only sample that fell in the desired absorbance range for 260 nm was sample A.
Therefore, sample A is the only sample that can be used to give an accurate concentrated of
isolated DNA. In order to determine the concentration of the isolated DNA, one must use Beer’s
Law and take into account that 1.0 ends up corresponding to a concentration of 50 ug/ml. The
calculations of DNA concentration is shown below.

Equation 2:
𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒
DNA concentration = 𝑚𝑜𝑙𝑎𝑟 𝑒𝑥𝑡𝑖𝑛𝑡𝑖𝑜𝑛 𝑐𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 ×𝑝𝑎𝑡ℎ 𝑙𝑒𝑛𝑔𝑡ℎ
0.150 𝐴 ×10 𝐷𝐹 1.0 𝐴 𝑢𝑔
= = 75 ⁄𝑚𝑙
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑥) 50 𝑢𝑔/𝑚𝑙

The yield of genomic DNA can then be found by using the average concentration of DNA and
multiplying it with the amount of water it was dissolved in, in this case 4 ml. The yield is the
amount of DNA in ug that was isolated.

Equation 3:
DNA Yield (ug) = DNA concentration x total sample volume (ml)
𝑢𝑔
DNA Yield = 75 ⁄𝑚𝑙 × 4 𝑚𝑙 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 = 300 𝑢𝑔
We can then convert the amount of genomic DNA from ug into grams.

1 × 10−6 𝑔𝑟𝑎𝑚𝑠
300𝑢𝑔 × = 3 × 10−4 𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝐷𝑁𝐴
𝑢𝑔

In order to find the experimental value of grams of DNA per bacterial cell, the total number of
cells must be calculated. In order to do so, the colonies grown on two separate LB plates were
4
counted in order to determine how many cells originated from each of the dilutions. The two
dilution factors used 105 𝑎𝑛𝑑 106 . Table 3 shows the results taken from each of the plates. Due
the fact that 105 had over 300 colonies, the more diluted plate, 106 , was used. One could have
anticipated that 105 would have more colonies present due to the fact that though the dilutions it
had ten times more the amount of bacteria than 106 . The results were close to ten times the
amount of bacteria in 105 plate than in 106 plate.

Table 3:
Total number of cells isolated from DNA
Dilution Factor
𝟏𝟎 𝟓
𝟏𝟎𝟔
Number of colonies 301 59

Equation 4:
59 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠
× 106 𝐷𝐹 × 75 𝑚𝑙 = 4.43 × 1010 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠
0.1 𝑚𝑙

Since it can be assumed that each colony originated from one cell, there was 4.43 × 1010 cells
present in the original culture. Now the experimental value can be determined using the mount of
cells originally present and the grams of isolated DNA.

Equation 5:
𝑚𝑎𝑠𝑠 𝑜𝑓 𝐷𝑁𝐴 (𝑔𝑟𝑎𝑚𝑠) 3.0 × 10−4 𝑔𝑟𝑎𝑚𝑠 𝑔𝑟𝑎𝑚𝑠
Experimental Value = = = 6.77 × 10−15 ⁄𝑐𝑒𝑙𝑙
𝑐𝑒𝑙𝑙 4.43 ×1010 𝑐𝑒𝑙𝑙

Discussion:

The theoretical value must be calculated in order to compare it to the experimental value and
ultimately find how accurate one was in DNA isolation. In order to find the theoretical value, one
must assume that E. coli chromosome contains 4 × 106 nucleotide pairs and the average
molecular weight of one nucleotide pair is 650𝑔⁄𝑚𝑜𝑙𝑒 . One must also take into account that one
mole is equivalent to 6.022 × 1023 molecules. With this knowledge, one can set up an equation
to find how many grams of genomic DNA is present in every cell.

Equation 6:

4 × 106 𝑛𝑢𝑐𝑙𝑒𝑜𝑡𝑖𝑑𝑒𝑠 650 𝑔𝑟𝑎𝑚𝑠 1 𝑚𝑜𝑙𝑒 𝑔𝑟𝑎𝑚𝑠

× × = 4.318 × 10−15 ⁄𝑐𝑒𝑙𝑙
1 𝑐𝑒𝑙𝑙 1 𝑚𝑜𝑙𝑒 6.022 × 1023

With both the theoretical and experimental value known, one can assess how accurate the DNA
isolation process was. The experimental value in this case was 6.77 × 10−15 𝑔𝑟𝑎𝑚𝑠⁄𝑐𝑒𝑙𝑙 which was
approximately two times larger than the calculated theoretical value, 4.318 × 10−15 𝑔𝑟𝑎𝑚𝑠⁄𝑐𝑒𝑙𝑙.
Since the experimental value was slightly greater than expected, there are many areas that could
have caused error in the experimental results. For example, although RNAse was used to degrade
RNA earlier in the experiment, there is still a chance that some of the RNA did not degrade. This
ultimately would increase the absorbance values since the spectrophotometer cannot differentiate
5
RNA with DNA. In order to determine the number of cells present in the samples of E. Coli
used, diluted amounts of the bacteria were plated. The plates were then grown overnight in order
to allow growth of colonies. The problem that arises is that many of the cells could have entered
the death phase of the growth curve. Since dead cells do not form colonies, the total number of
colonies could have been significantly less than the actual amount of cells present in the bacteria.
Ultimately, the dead cells would affect the apparent cell density by decreasing its value and
increasing the DNA/cell calculation.
There is also the possibility that the source of error decreased the amount of isolated DNA
measured. This can occur when transferring the DNA from the glass rod to the tube. This method
is not a very precise way to transfer all of the DNA. Some of the DNA could have never been
attached to the glob and could have remained in the aqueous solution. Another potential area of
error is when adding ethanol to DNA. Some of the DNA could have remained in the solution
instead of precipitating out. This can be resolved by better mixing of the two substances. With
the many different variables that can cause discrepancies in the experimental values, it is
important to do genomic DNA isolation and quantification as accurate as possible and to be
aware of areas where errors may arise.

References:
Alberts, Bruce. "Molecular Biology of the Cell. 4th Edition." Isolating, Cloning, and Sequencing
DNA. U.S. National Library of Medicine. Web. 9 Sept. 2015.
<http://www.ncbi.nlm.nih.gov/books/NBK26837/>.

Smith, Cassandra L., and Linda G. Tolstoi. "Genomics." - Biology Encyclopedia. Web. 9 Sept.
2015. <http://www.biologyreference.com/Fo-Gr/Genomics.html>.