plasmid dna: extraction, restriction mapping, and sequencing

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Christina Cubillo & Siavosh Naji-Talakar Lab Exercise B1, B2, and B3 Lab Section: Tuesday 3 P.M. Andy Diamos Plasmid DNA: Extraction, Restriction Mapping, and Sequencing

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Christina Cubillo & Siavosh Naji-Talakar

Lab Exercise B1, B2, and B3

Lab Section: Tuesday 3 P.M.

Andy Diamos

Plasmid DNA: Extraction, Restriction Mapping, and Sequencing

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Introduction

Bacterial plasmids are an extra-chromosomal circular piece of DNA that usually have

three key elements beginning with their origin of replication, a selectable marker gene, and a

cloning site (Metzenberg). The origin of replication (ori) is where specific genes are encoded that

pertain to plasmid replication. Selectable markers, often antibiotics, are used to give the

recombinant plasmid a way to be identified after it is incubated. Cloning sites are areas where

restriction enzymes act and may be used to insert DNA fragments. Plasmid vectors are used

extensively in recombinant DNA techniques. These plasmid vectors may be used to clone,

propagate, manipulate and deliver any specific DNA sequence or even to express proteins

(Mason and Mor, 2015). Once DNA is extracted and prepped the Beer-Lambert law, which

measures light absorbance at 260nm to 280nm, may be applied to evaluate the purity of the

extracted DNA. Restriction enzyme can be added to DNA to cut plasmids at specific points.

These restriction enzyme in turn can be used to make a restriction map of the DNA sequences

cut by electrophoresis.

In order to extract plasmids from bacteria a preparation method must be used. The

preparation steps are used in order to lyse the bacterial cell and allow the cell contents, which

includes the plasmid, to be sorted out from contaminants such as proteins, RNA, or

carbohydrates. There are different methods to extract plasmid DNA however since it has become

a common practice “miniprep” kits are available that use the alkaline lysis method. Alkaline lysis

begins by first taking bacterial cells from E. coli that contain a plasmid that is to be isolated. The

solution is spun down to create a pellet surrounded supernatant. The supernatant in discarded

since pieces of cell wall are released which may inhibit enzyme action on DNA (Guruatma,

2010). The harvested bacteria cell is re-suspended in a Tris-Cl which acts as a buffer, ethylene

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diamine tetraacetic acid (EDTA) that acts as a chelator further weakening the cell wall and

ribonuclease A that hydrolyzes and contaminating RNA (Mason and Mor, 2015). Sodium

dodecyl laurylsulfate (SDS) acts as a detergent and pops holes in the cell membrane combined

with sodium hydroxide (NaOH) that loosens the cell walls and releases the plasmid DNA which

are used to lyse the cells (Guruatma, 2010). The plasmid DNA is renatured with the addition of

potassium acetate (KAc) and centrifuged to remove all debris. The plasmid DNA is now in the

supernatant while all the contaminants are in the pellet (Guruatma, 2010). The extracted DNA is

further purified by binding it under high chaotropic salt conditions to silica. The proteins or other

contaminants may then be washed away and the DNA is finally eluded with low-salt solution.

The concentration of DNA in a solution may be understood by using the Beer-Lambert

law which draws a correlation between absorbance and concentration (Watts, 2014). DNA has a

peak absorbance of 260nm. Nucleic acids absorb UV light at the 260nm because of the aromatic

base moieties located in their structure and can be applied to Purines and pyrimidines (Watts,

2014). Proteins and other phenolic compounds have a stronger absorbance of 280nm. The ratio

of A260/280 is used to determine the contamination of DNA samples by proteins. The ratio for a

pure sample of DNA should be around 2.1 and 1.8 with a lower ratio indicating the sample is

contaminated with proteins (Watts, 2014).

Restriction enzymes are any enzyme that recognizes and cleaves a specific short

sequence on the restriction site in double stranded DNA; this method is used to produce

recombinant DNA in vitro and is also called restriction endonuclease (Lodish et. al, 2013).

Bacteria typically use restriction enzymes in order to defend against bacteriophage DNA

attacking their own DNA. These enzymes are palindromic and typically recognize a specific 4 to

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8bp sequence, restriction site, and cleave the DNA strand leaving either blunt ends or overhangs.

Bacteria protect their own DNA from restriction enzymes by adding a methyl group allow the

bacteria to recognize internal DNA from external. To perform a restriction endonuclease digest

DNA is incubated with the enzyme. Digest solution includes the plasmid DNA, any choice of

restriction enzymes and buffer solution consisting of magnesium ions, sodium or potassium salts,

Tris buffer, dithithreitol and bovine serum albumin (BSA) (Mason and Mor, 2015). The entire

solution is typically incubated at 37°C.

Agarose gel electrophoresis is a method that can be applied to separate DNA fragments

based on their size. This method is especially applicable to DNA cut with restriction enzymes

which can help to visualize each cut made in the original DNA. Electricity is run through the gel

with DNA samples loaded into each “well” within the gel. The negative charge of the DNA

phosphate backbone are drawn towards the positive end of the gel electric field. The smaller

DNA move further down the electric current. As the electric current moves the agarose acts as a

kind of sieve that filters smaller to larger DNA. Ethidium bromide is an intercalating dye that can

insert itself between the bases of the stacked DNA helix (Guruatma, 2010). The finished gel

may be viewed under UV light to indicate the size of each DNA strand compared known tree.

Thus, the length of each DNA strand may be determined by analyzing the gel and determination

of the way each restriction enzyme cuts and at what bit-point (bp) on the DNA may be inferred.

Adding this data together can determine the length of the original plasmid prior to any cutting.

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Materials and Methods

Lab B1:

Qiagene Alkaline Lysis Miniprep-

Two preps were prepared involving the extraction and purification of plasmid DNA from

cultures of E. coli. The E. coli was equipped by using a single well- isolated colony from an LB

agar plate to inoculate 1-5 mL of LB medium. Subsequently, the E. coli was incubated overnight

at 37℃ for 12-16 hours. A cleared lysate was manufactured by harvesting 1-5 ml of plasmid by

centrifugation; the supernatant was then poured into the designated flask containing bleach.

Pelleted cells were suspended in 250µl of Buffer P1 by vortexing. 250µl of Lysis Buffer P2 was

added and inverted 4-6 times to mix. 350µl of Buffer N3 was added and inverted immediately 4-

6 times. A compact white pellet was formed after the substance was centrifuged for 10 minutes.

The isolation and distillation of plasmid DNA was achieved by applying supernatants to

the QIAprep spin column by pipetting and centrifuging for 30-60 seconds. The spin column was

washed by incorporating 0.75 ml of Wash Buffer PE and centrifuging for 30-60 seconds. The

flow-through was discarded and centrifuged for an additional minute to remove residual wash

buffer. The spin column was placed in a sterile 1.5 ml micro centrifuge tube. The DNA was

eluted by adding 50µl of ddH2O to the midpoint of each spin column; allowed to sit for 1

minute, then centrifuged for 1 minute.

Detection of Nucleic Acids Using Absorption Spectroscopy-

The concentration of the A260/A280reading was determined in each prep by diluting the

sample 1:60 in 120 µl of ddH2O. A spectrophotometer was used and set to fixed wavelength

mode; wavelengths were adjusted to the appropriate measurements that read 260nm and 280nm.

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The apparatus was zeroed with water followed by distributing the sample into a cuvette. DNA

concentration was calculated using ɛ=0.02(µg/ml)-1cm-1. A260/A280was calculated.

The concentration of the 200nm-300nm scan was established by the same methods

described in the A260/A280reading procedure. The only dissimilarity is that the

spectrophotometer was set to scan mode while the wavelengths were altered to read 200nm-

300nm.

Lab B2:

Restriction Digest-

The plasmid prepared from the previous lab was digested with restriction enzymes; singly

using NcoI and in combination utilizing NcoI and SacI. Sterile micro centrifuge tubes were used

to place the restriction digest. The single digest consisted of: 0.5µl NcoI, 12.5µl sterile ddH2O,

5µl DNA, and 2µl buffer 4 cutsmart. The combination digest consisted of: 0.5 µl NcoI and Sac I,

12µl sterile ddH2O, 5µl DNA, and 2µl buffer 4 cutsmart.

Lab B3:

Resolving DNA fragments by agarose gel electrophoresis-

Agarose electrophoresis was used to resolve the restriction fragments produced by the

digestion in Lab B2. 500 ml of TAE buffer was prepared along with the preparation of 1.2%

agarose gel. The solution was microwaved for 1 minute then mixed to ensure all agarose was

dissolved. The agarose was cooled then incorporated into the middle electrophoresis chamber.

The comb was inserted to the side closest to the negative charge and was left untended for 10-20

minutes so the gel may solidify.

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Once the agarose was hardened in the electrophoresis chamber it was immersed in a

electrophoresis buffer. The comb was then gently pulled out. The wells were loaded with 5µl of

each sample by pipetting. The samples that were inserted into the gel was prepared by adding

loading dye; so the fragments will be visible under a UV light. The samples were arranged

involving 1-kb ladder standards.

The electrophoresis box was then covered followed by turning on and adjusting the

power supply to 70 V. The gel ran for 60-80 minutes. The gel from the gel electrophoresis platter

was removed and placed into a tray that contains ethidium bromide. The tray was positioned onto

a shaker and stained for 10 minutes. The gel was destained by aligning it onto a tray containing

distilled water. A UV source was used to view the gel. The motilities of standards and samples

were measured as well as fragment sizes.

Results

The first step of the experiment required for the previously inoculated LB agar plate

medium containing E. coli colony to be used in order to inoculate 50ml of

LB broth. The LB was successfully inoculated and incubated from the

previous lab session and used. We transferred 2.0ml of this culture and

centrifuged at 10,000rpm for 1 minute. Observations were made that the

heavier cell particles were forced to the bottom of the tube. The

supernatant that remained was carefully removed via pipette tool as to

ensure the pellet at the bottom was not disturbed. The pellet was then

re-suspended in 250ul of buffer P1 for 1 minute. It was important to

ensure that there was no clumps of the pellet remaining stuck at the

bottom of the tube. We ensured to break up all the clumps then vortexed

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Image 1 - Pellet after removal of supernatant

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the tube again just to be very sure the entire pellet was completely broken up and in full contact

with the buffer. After the vortex 250ul of lysis buffer (P2) was added. In this step it was

important for us to not vigorously shake the tube because that would lead to star activity.

Therefore, to mix the P2 buffer we simply inverted the tube slowly 5 times. The final buffer N3,

350ul, was then added. It was critical to mix this buffer immediately so we

would avoid localized precipitation by inverting the tube gently 10 times.

The solution now was observed to be very white and cloudy. The entire

solution containing the buffers and culture were then centrifuged for 10

minutes.

After the 10 minutes of centrifuge was complete the DNA was

successfully lysed from the cell. Since the DNA weighs less than the remaining proteins and

other cell particles (contaminants) it is now located in the supernatant. The supernatant was

carefully transferred to a QIAprep spin column by pipetting. The column was centrifuged for 1

minute and the flow through discarded. The DNA is now in the column attached to the silica. We

observed a lot of flow through on the initial centrifuge. Therefore the QIAprep column was

centrifuged again to fully dry the spin column and a minuscule amount

of liquid was left as the flow through. This indicated that the column is

properly dry.

The final step was to elute the DNA from the column, dissociate

it from the silica, by adding ddH20. The blue column of the QIAprep

was put on top of a new 1.5ml tube. Using a pipette tool 50ul of ddH20

was added to the center of the spin column. The column was let to stand

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Image 3 - QIAprep column with flow through

Image 4 - DNA eluded from column to 1.5ml tube

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for 1 minute then centrifuged for 1 minute with the cap of the 1.5ml tube open. The column was

discarded as we successfully transferred our sample DNA to the bottom of the 1.5ml tube.

Now that we have successfully attained the purest sample of DNA from our

culture we may measure the concentration and purity using absorption

spectroscopy. A 1/25 dilution of plasmid DNA was first made by mixing 3ul with

72ul of ddH20. The remaining undiluted plasma DNA was stored in the freezer for

later use. The absorption spectroscopy machine uses cuvette to hold material for

sampling. We ensured to wash the cuvette with distilled water then add 50ul of distilled water to

“zero” the machine. For our official test we transferred 50ul from

the 1/25 diluted 75ul to the cuvette. Absorption spectroscopy was

run and found to be 54.1 ng/ul total with a 260/280 ratio of 1.54

and a 260/230 ratio of 2.32.

Taking our purified sample of DNA with identified

concentration allows us to begin the restriction endonuclease of the plasmids

contain in the sample. NcoI and SacI were the restriction enzymes chosen to cut

the plasmid. NcoI and SacI were chosen because they both work at 100% activity

in buffer solution 4. We used two 1.5ml tubes. The first was a NcoI enzyme

only and added to it 5ul of DNA, 2ul of the restriction buffer 4,

0.5ul of the enzyme NcoI, and 12.5ul of ddH20 to fill to 20ul total volume. The

second was NcoI + SacI enzymes double cut which were added to 5ul of DNA, 2ul of

the restriction buffer 4, 0.5ul of the enzyme NcoI, 0.5ul of the enzyme SacI and

12.0ul of ddH20 to fill to 20ul total volume. There was no visible change or

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Image 5 - Cuvette

Image 6 - Absorption Spectroscopy

Image 7 - Restriction

Buffer

Image 6 - DNA solution with

restriction enzyme and buffer

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occurrence of any abnormalities within the substrate. The tubes were briefly spun down on a

centrifuge and then incubate to allow the restriction enzymes to work.

Table 1 % activity in NEB buffersEnzymes Recognition

SequenceRecom.Buffer

Recom.Temp

1 2 3 4

NcoI C/CATGG 4 100 100 100 100SacI GAGCT/C 1+BSA 100 50 10 100

The restriction enzymes were given adequate time to inoculate and finish their processing

of the DNA plasmid. In order to visibly see what the results of the experiment were gel

electrophoresis was necessary. The gel used for the electrophoresis had to first be manufactured.

Initially 490ml of ddH20 was mixed with 10ml TAE. TAE is a mixture of Tris-acetate pH8.0 and

50mM EDTA). We took 40ml of this solution and added it to a separate 125ml flask then added

1% w/v of agarose which was about 0.418g. This flask was microwaved for about a minute or

until we observed boiling bubbles. Gel solution with agarose in the flask was allowed to cool to

room temperature then cast into the plastic gel

holder area of the electrophoresis machine. The

comb, which creates the wells for DNA samples,

was added on top of the gel to create the wells as

the gel hardened. It was important to ensure the

wells were created on the black negative head

side of the machine so the DNA would flow across the gel to the positive red terminal. After the

gel had hardened the comb was removed exposing the sink holes and remaining 460ml of

solution was poured all over and across the gel membrane and allowed to fill the wells on each

side.

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Image 8 - Gel Electrophoresis in progress with loaded DNA

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The samples containing DNA that were cut with restriction endonucleases were then

prepared with a loading dye. 20ul of the DNA sample was combined with 2ul of loading dye,

vortexed, and spun down. Using a micropipette tool 15ul of the DNA mixed with loading dye

was very carefully pipetted into the sink holes in the dye. The

NcoI sample was loaded first on the far left of the gel and the

NcoI + SacI sample were loaded to the right. The 1 kb ladder

was loaded to the far left side of the gel. The electrophoresis

machine was run for one hour. Once complete the gel box

was removed and placed under a UV light to allow the

fluorescent dye to glow. Observations were made and photos

taken.

Discussion

In all experiments complete and through each step proper aseptic technique was adhered

to and it may be assumed that no contamination from outside DNA contributed to the results

attained. The stock of E. coli used was previously incubated from a fresh starter culture provided

by the lab assistant. Experiment B1 required the lysing of the E. coli cell in order for its DNA to

be released and collected. The initial results of the purified DNA using the spectroscopy showed

a very low concentration of around 1.2 A260/280. We were puzzled as to why we had such a low

reading so a second pass through the machine was made with a few adjustments. The second

time we ensured that the machine was first calibrated with a cuvette and the same cuvette was

used to add our DNA sample into. However, the second result indicated a still low 1.54

A260/280 concentration. This means that there were contaminants in the DNA. Identifying the

issue of why there was such a low concentration was perplexing at first since we ensured to

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Image 9 - Gel Electrophoresis results

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follow the steps very closely. After careful consideration it was concluded that after the lysis

buffers were added to the original DNA sample the tube was not inverted in a gentle manner.

This slightly vigorous shaking of the lysis buffers mixed too much with the cell debris and

caused our contamination. This low concentration DNA did not yield any results when put under

the UV light after electrophoresis and therefore we were forced to use another group’s data.

Adding the restriction enzymes to the purified DNA was considered and easy and trivial step.

We may have encountered errors though in our measurements with the pipette. The accuracy of

the restriction enzyme amounts mixed with the DNA samples may not be on point and thus

caused errors in our final gel electrophoresis. With the creation of the gel for the gel

electrophoresis we also experienced an errors. Several groups did not properly mix the ddH20

with TAE first before taking 40ml and combining with agarose. We were successful in this step

however we failed to insert our comb into the gel immediately. The delay in inserting the comb

caused our gel to rip apart when the comb was removed and rendering our gel unusable. Our

group was forced to use another group’s electrophoresis gel and machine.

The data gathered from successful experiments include results on NcoI, NcoI + SacI, and

EcoRI restriction enzymes from other groups gel electrophoresis. NcoI, based on electrophoreses

gel results, is indicated to cut our E. coli plasmid at 3000bp. When NcoI and SacI are used in a

double digest it can be seen that NcoI cuts still at 3000bp and now SacI cuts at 500bp. Reviewing

results from EcoRI we see that the DNA remained supercoiled therefore it was not cut at all.

Summarizing all of this information allows us to deduce that the entire length of our DNA

plasmid is 3500bp and can be successfully cut with NcoI and SacI but not with EcoRI.

Restriction mapping of the sequence us as follows:

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Restriction endonucleases with gel electrophoresis have many real world applications that

span across many fields. One of the prime example of electrophoresis impacting us daily is in the

field of forensics. Gel electrophoresis is used to obtain DNA fingerprints of criminals or victims.

Scientist can tell the difference between two pieces of DNA from a crime scene up to a very high

margin of accuracy. In the field of molecular biology we use the technique to separate DNA or

RNA and organize them by size which can make it much easier to study on a molecular level.

Genetics uses the technique to prepare their DNA samples to be cloned or genetically

engineered. Microbiologist apply the technique in the field of virology to help diagnose different

viruses. Lastly, electrophoresis is used in biochemistry where biochemists work with many

different cell parts such a proteins or nucleic acids. They can map these different parts using

electrophoresis.

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Image 10 - Restriction map of sample plasmid

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References

Guruatma, J. (2010, April 12). Mama Ji's Molecular Kitchen. ASU - Ask A Biologist.

Retrieved September 27, 2015, from https://askabiologist.asu.edu/alkaline-lysis

Guruatma, J. (2010, April 2). Mama Ji's Molecular Kitchen. ASU - Ask A Biologist. Retrieved

September 26, 2015 from http://askabiologist.asu.edu/agarose-gel-electrophoresis

Lodish et al. Molecular Cell Biology 7th Edition. (2013).New York: W.H. Freeman and

Company.

Mason, H, Mor, T. 2015. Lab Exercise A1 Working with Bacteria-the basics; Lab Exercise A2

Working with Bacteria-obtaining pure cultures. Arizona State University, Tempe, AZ.

Metzenberg, S. (n.d.). Bacterial Plasmids. Retrieved September 27, 2015, from

https://www.csun.edu/~hcbio027/biotechnology/lec2/PL/pl.htm

Watts, G. (2014, August 19). Interpreting Nanodrop (Spectrophotometric) Results. Retrieved

September 27, 2015, from http://www.u.arizona.edu/~gwatts/azcc/InterpretingSpec.pdf

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