c






 

Taness Billington

Mic 428L/ Section 003

Dr. Cooper/Melissa Valdez

May 4th, 2011

c



The goal of this experiment was to isolate chromosomal DNA from a couple of our

mutants in experiment 5 and digest with a restriction enzyme. The fragments will then be self

ligated and transformed into a strain of E. Coli DH5ĮȜë
containing the ë
gene ʌ product for

replication. This will allow only the plasmid containing the Tn to replicate. We will then isolate

the plasmid and sequence it into adjacent chromosomal DNA to determine which gene was

interrupted by the Tn.

The isolation of genomic DNA is a prerequisite for many types of experiments including

DNA sequencing and cloning. One common goal of the isolation of the chromosomal DNA is to

avoid shearing. The large chromosome is fragile and can be sheared very easily. Although there

will always be some chromosomal DNA that cannot avoid shearing, most methods use a gentle

lysis step using a detergent, SDS, and a proteinase enzyme to degrade proteins. Chloroform will

also be used in the isolation procedure to remove any additional proteins bound to the DNA that

we do not want. Additionally, the chromosomal DNA will precipitate out using a non polar

solvent, isopropanol or ethanol. DNA is polar because of the negative charge on its phosphate

backbone. Therefore, when it is exposed to a less polar solvent, such as ethanol or isopropanol, it

will reduce the amount of surface area that is exposed to that solvent therefore it will form a ball

and precipitate out. The kit we will be using for our isolation is the Gentra Puregene Yeast/Bact.

Kit from Qiagen, Inc. as it is very quick and produces quality chromosomal DNA for digestion

with restriction enzymes.

The chromosomal DNA will be digested with a restriction enzyme once it has been

isolated. We will use the restriction enzyme BamHI which cuts in fragments of around 5,000

base pairs. This means that BamHI will digest the chromosome once every 5,000 base pairs. For
a genome of 6 Mb, BamHI will digest it into 1,200 fragments. One of our goals of this

experiment was to self ligate these fragments. The idea behind this is that only one of these self

ligated fragments will contain the Tn, therefore, if we transform all of them into E. Coli, only the

circular piece containing the Tn with the ʌ dependent R6 origin of replication will replicate.

Many bacteria in nature are able to naturally take up exogenous DNA from the

environment. E. Coli, the bacteria that we are working with, however, does not, and needs to

made competent in order to take up the exogenous DNA by electroporation. In this procedure, E.

Coli is made competent by adding CaCl2, and glycerol. To transform the DNA into the

competent E.Coli, electroporation is used. Electroporation is an increase in electrical

conductivity and membrane permeability as a result of an applied electric field.

` 

 

Purification of Genomic DNA:

An overnight culture of the mutants selected is prepared and transferred to a 1.5 ml

microfuge tube on ice. The cells are pelleted by centrifugation. The supernatant is discarded and

Cell Lysis solution is added and mixed. Cell lysis solution is a detergent that is used to lyse the

cells and avoids shearing the DNA. RNase A solution is then added to remove any additional

proteins. Protein precipitation solution is then added to allow for the chromosomal DNA to

precipitate out. Isopropanol is then added to the previous supernatant. Isopropanol is a non polar

solvent. DNA is very polar therefore when exposed to a polar solvent it will reduce its surface

area exposed to the solvent and will collapse into a ball. The DNA should be visible as a small,

white, pellet. Ethanol is then added to wash the DNA pellet of any remaining contaminants. The

supernatant is then discarded, and the tube is turned upside down, making sure the pellet remains
inside. Hydration solution is then added and the mix is vortex in order to break apart the pellet.

The sample is then incubated at room temperature overnight with gently shaking.

Chromosomal Digestion

Our isolated chromosomal DNA from the previous step with them be mixed in with BamHI in

order to digest it into many small fragments. They are then incubated and allowed to digest in a

water bath for 2 hours.

Ligations

The restricted digested chromosomal DNA will then be mixed with ligase solution so that DNA

ligase can self ligate the fragments into circles. A ligase buffer and water is also added to this

solution. They are then incubated overnight and stored until the next lab period.

Transformation by Electroporation

2ȝl of the ligation mix will be added to a 1.5ml chilled tube of electrocompetent EC1000 ë
-

116, which is the strain of  that has already been made competent for us. 22ȝl will then be

added to a chilled electroporation cuvette. The cells will be electroporated using a Bio-Rad Gene

Pulser, which will send electrical pulses through the  so that the exogenous DNA can enter

it. Room temperature SOC buffer is then added to stabilize the cells after being in a state of

stress from the electroporation. The solution is then transferred to a new 1.5 ml microfuge tube

and stored at 37°C for 60 minutes. 200ȝl of the solution will then be spread on an LB+Kan50

plate to select for the plasmid that contains the kanamycin resistance marker. These plates will be

incubated at 37°C for 1-3 days. After 1-3 days, we will select 2 colonies from each plate and

retouch a new LB+Kan50plate in order to ensure that we obtained the plasmid DNA.
Isolation of plasmid DNA for sizing and sequencing

The isolated plasmid DNA will then be run on an agarose gel to determine the size of it.

Tracking dye will be added to the solution which contains glycerol that will weigh the samples

down in the wells.

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Figure 1 shows our fragment that was run through gel electrophoresis. Our fragment is seen here

in well 7 and 8. The Kb ladder can be seen in well 1.

  

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