E coli - LAB REPORT ON AGAROSE GEL ELECTROPHORESIS PDF

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Summary

LAB REPORT ON AGAROSE GEL ELECTROPHORESIS...

Description

Objectives 

To purify a restriction endonuclease



To test the enzyme activity of the restriction endonuclease



To visualize the test results by agarose gel electrophoresis

Introduction A commonly used protein purification method that is both efficient and reliable is column chromatography. In this method, the column (a glass or plastic cylinder) is packed with a solid matrix and it has an aqueous buffer reservoir at the top and a fraction-collecting device at the bottom. A buffered solution containing a protein extract is added to the top of the column by gravity or peristaltic pumping. Once the solution has entered the matrix, the same buffer without protein, sometimes called running buffer, is continually added to the column. Based on differential physical or chemical interactions between the proteins and the column matrix, subsets of proteins in the mixture exit the column at different times. Ion-exchange Chromatography is a type of column chromatography that exploits charge differences between proteins. Net positively charged or net negatively charged proteins are bound differentially, depending on the charge on the column matrix and the pH of the buffer. Two commonly used ion-exchange matrices are a positively charged anion-exchange matrix called diethylaminoethyl

(DEAE)

cellulose

and

a

negatively

charged

cation-exchange

matrix

called

carboxymethylcellulose. An anion-exchange resin binds negatively charged proteins (anions), whereas a cationexchange resin binds positively charged proteins (cations). Once the protein sample is loaded onto the column, the oppositely charged protein and resin interact, and the matrix is extensively washed with loading buffer to remove unbound protein. An elution buffer is then added to the column: This buffer contains a high concentration of an appropriate competing ion such as Na+ or Cl− that displaces the bound protein. The eluted fractions are assayed for the target protein, then pooled and dialyzed for further use. Sequence-specific, or type II, endonucleases are commonly known as restriction enzymes. In contrast with nonspecific endonucleases, restriction enzymes generate reproducible fragments from specific DNAs. They cleave 1

double-stranded DNA by hydrolyzing two phosphodiester bonds (one per strand) within defines nucleotide sequence. The name of a restriction enzyme is derived from the genius and species of bacterium from which it is isolated.

Materials/Reagents 

Distilled water



Agarose powder



SYBR® Safe Stain



Eco RI



50x electrophoresis Buffer



Lambda DNA



KCl solutions



DEAE-Cellulose

Experimental Procedure Parking and equilibrating the column 

The column was vertically mount on a ring stand.



The cap was slided onto the spout at the bottom of the column.



1.5mL of DEAE-Cellulose (Ion exchange matrix) was mixed thoroughly by stirring and gently pipetted slowly down the inside walls of the column.



The packed column was washed with 6mL of eq (1x equilibration buffer)

Collecting column fractions 

Nine test tubes were labelled #1-9



1mL of distilled water was added to test tube #1 and used as a reference for collecting fractions.

2



The column was slowly loaded with 1mL of E. coli RY extract. The cap was removed to allow the extract to completely enter the column matrix.



2mL of the equilibrating buffer was slowly added to the column to remove protein that is in the flow through.



Two fractions each of 1ml were collected into test tubes 2 and 3. The fractions were immediately stored in ice.



The column was slowly eluted with 2mL of 0.1M KCl and two fractions each of 1mL were collected into test tubes 4 and 5.



The column was slowly eluted with 2mL of 0.2M KCl and two fractions each of 1mL were collected into test tubes 6 and 7.



The column was slowly eluted with 2mL of 0.5M KCl and two fractions each of 1mL were collected into test tubes 8and 9. All the fractions collected were stored in ice.

Analysis of Eco RI Activity (First assay) 

Nine micro centrifuge tubes were labelled #1-9



Assigned amount of qualified water was added to each test tube.



5µL of Eco RI Rxn buffer and 5µL of Lambda DNA were added to each of the tubes.



Assigned fractions were added to the tubes. Remaining amount of each fraction was stored in a refrigerator for later use.



The tubes were capped, and samples were collected at the bottom of the tubes by tapping or spinning.



The tubes were incubated over a water bath at 37oC for about 15mins.



After incubations, 5µL of 10x gel loading solution was added to each tube to stop the reactions. This prepares the Eco RI digestion products for separation by agarose gel electrophoresis.

Preparation of Agarose gel with SYBR® Safe Stain 

50x electrophoresis buffer was diluted with distilled water to a 1x buffer.

3



The agarose powder was mixed with the 1x buffer in a 250ml volumetric flask. The mixture was boiled and then microwaved to ensure total dissolution of the agarose powder in the buffer. A clear solution was observed.



The agarose solution was cooled to 60oC by swirling to ensure even dissipation of heat.



The ends of the gel-casting tray was sealed with rubber end caps.



Diluted SYBR® Safe Stain was added to the cooled molten agarose and swirled to mix.



The cooled agarose solution was poured onto the prepared gel-casting tray. The gel solidified after 20mins and become less transparent.



The end caps and comb were slowly and gently removed.



The gel on the tray was placed into the electrophoresis chamber. The gel is covered with 1x electrophoresis buffer to completely submerge the gel.



20µL of each sample was loaded in the wells in order. The gel was properly oriented, and the safety cover was placed onto the chamber. DNA samples migrate towards the positive electrode.



The leads were connected to a power source to commence electrophoresis. After the process was complete, the gel was removed from the casting tray and visualized.

Quantification of Eco RI activity (second Assay) 

Six micro test tubes were labelled (0, 1:2,1:3, 1:4, 1:10 and 1:20). Seven other micro test tubes were also labelled #1-7.



From the agarose gel results, the two fractions with the highest Eco RI activity were determined. More activity were the ones with more digestion of plasmids.



The two fractions were combined into a 5mL snap-top tube, capped and gently inverted to mix. This is the pooled enzyme fraction.



Using the tubes labelled 0, 1:2,1:3,1:4,1:10, and 1:20, Eco RI initial dilutions were prepared according to table 4.

4

The Eco RI initial dilutions from the previous step was used to prepare assay dilutions in tubes labelled



1-7 from table 5. 

Tubes 1-7 were incubated in a water bath at 37oC for 30mins.



5µL of 10x gel loading solutions was added to each tube 1-7 to stop the reactions. This prepares the Eco RI digestion products for separation by agarose gel electrophoresis.

Data/Results TABLE 1: Key for identifying fractions Tube 1

Fraction 1mL reference guide

2

(no salt)

3

(no salt) 0.1M KCl

5

0.1M KCl

6

0.2M KCl

7

0.2M KCl

8

0.5M KCl

9

0.5M KCl

Rxn Tube 1

TABLE 3: Sequence for Restriction Enzyme Reactions 37oC Qualified Eco RI Lambda Fraction Reaction water reaction DNA (Amount volume incubation (µL) buffer(µL) (µL) assigned) (µL) (minutes) 40 5 5 None 50 15

2

(40-x)

5

5

3

(40-x)

5

5

4

(40-x)

5

5

5

(40-x)

5

5

x µL tube 2 (No salt) x µL tube 3 (No salt) x µL tube 4 (0.1M KCl) x µL tube 5

10x Gel load (µL) 5

50

15

5

50

15

5

50

15

5

50

15

5 5

6

(40-x)

5

5

7

(40-x)

5

5

8

(40-x)

5

5

9

(40-x)

5

5

(0.1M KCl) x µL tube 6 (0.2M KCl) x µL tube 7 (0.2M KCl) x µL tube 8 (0.5M KCl) x µL tube 9 (0.5M KCl)

50

15

5

50

15

5

50

15

5

50

15

5

Gel Loading Table – First Assay TUBE SAMPLE Marker Lambda Eco RI Marker 1 Uncut Lambda DNA 2 Lambda + fraction 2 (no salt) 3 Lambda + fraction 3 (no salt) 4 Lambda + fraction 4 (0.1M KCl) 5 Lambda + fraction 5 (0.1M KCl) 6 Lambda + fraction 6 (0.2M KCl) 7 Lambda + fraction 7 (0.2M KCl) 8 Lambda + fraction 8 (0.5M KCl) 9 Lambda + fraction 9 (0.5M KCl)

LANE 1 2 3 4 5 6 7 8 9 10

TABLE 4: Dilution of Pooled Eco RI Dilution Factor Pooled enzyme fraction

Eco RI dilution Total Buffer Volume

0

10 µL

0 µL

10 µL

1:2

5 µL

5 µL

10 µL

1:3

10 µL

20 µL

30 µL

1:4

10 µL

30 µL

40 µL

1:10

10 µL

90 µL

100 µL

1:20

5 µL

95 µL

100 µL

6

TABLE 5: Assay to Determine Total Units of Eco RI Lambda DNA

Reaction Volume

40 µL

Eco RI reaction Buffer 5 µL

5 µL

50 µL

10 µL of 0

30 µL

5 µL

5 µL

50 µL

3

10 µL of 1:2

30 µL

5 µL

5 µL

50 µL

4

10 µL of 1:3

30 µL

5 µL

5 µL

50 µL

5

10 µL of 1:4

30 µL

5 µL

5 µL

50 µL

6

10 µL 1:10

30 µL

5 µL

5 µL

50 µL

7

10 µL of 1:20

30 µL

5 µL

5 µL

50 µL

Rxn Tube #

Eco RI dilution (From table 4)

Qualifie d water

1

None

2

7

Conclusion Agarose gel electrophoresis has proven to be an efficient and effective way of separating nucleic acids. Agarose's high gel strength allows for the handling of low percentage gels for the separation of large DNA fragments. Molecular sieving is determined by the size of pores generated by the bundles of agarose in the gel matrix. In general, the higher the concentration of agarose, the smaller the pore size. Traditional agarose gels are most effective at the separation of DNA fragments between 100 bp and 25 kb. To separate DNA fragments larger than 25 kb, one will need to use pulse field gel electrophoresis, which involves the application of alternating current from two different directions. In this way larger sized DNA fragments are separated by the speed at which they reorient themselves with the changes in current direction. DNA fragments smaller than 100 bp are more effectively separated using polyacrylamide gel electrophoresis. Unlike agarose gels, the polyacrylamide gel matrix is formed through a free radical driven chemical reaction. These thinner gels are of higher concentration, are run vertically and have better resolution. In modern DNA sequencing capillary electrophoresis is used, whereby capillary tubes are filled with a gel matrix. The use of capillary tubes allows for the application of high voltages, thereby enabling the separation of DNA fragments (and the determination of DNA sequence) quickly. Once the DNA have been separated it can be viewed by placing the gel under UV light in a Gel Doc machine, due to the presence of dye the bands of DNA fragments glow. Fractions 6 and 7 have EcoR1 fragments after partial purification as most of the lighter bands generated have same size as that of the marker. The peak activity of EcoR1 could be seen in fraction 6 as maximum fragments are generated. Analyzing the data, it can be interpreted that fractions1, 2 and 3 have similar sized fragments of DNA, therefore action of the enzyme in the presence of 0.1M salt and no salt are similar. For fraction 4, the fragments are seen to overlap due to similar size, the same is in case of fraction 8 and 9, and the separated 8

fragments are seen as overlapping bands. In case of fraction 6 and 7, the fragment size is similar and maximum similarity of the fragment size can be found when compared to the marker. The recognition site for Eco RI enzyme is 5' GAATTC 3 3' CTTAAG5 The restriction endonuclease EcoRV (from E. coli) cleaves double-stranded viral DNA molecules that contain the sequence 5′-GATATC-3′ but leaves intact host DNA containing hundreds of such sequences. The host DNA is protected by other enzymes called methylases, which methylate adenine bases within host recognition sequences. For each restriction endonuclease, the host cell produces a corresponding methylase that marks the host DNA and prevents its degradation. These pairs of enzymes are referred to as restriction-modification systems. The Lambda DNA used in this experiment is isolated as a linear molecule from the E. coli bacteriophage lambda. It contains approximately 49,000 base pairs and has 5 recognition sites for Eco RI, and 7 for Hind III. Total activity is the amount of enzyme activity recovered from the preparation. It does not indicate the level of enzyme purity. Specific activity on the other hand is the amount of enzyme units per mg of total protein in the enzyme fraction. The less total protein the Eco RI fraction contains, the higher its specific activity.

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