Restriction Digestion And Gel Electrophoresis Biology Essay

The experiment was to test out which bacterial civilization was successfully cloned with the recombinant DNA insert. The methods involved the usage of commercial kit, QIAprep Spin Miniprep to pull out and sublimate the plasmid Deoxyribonucleic acid from the bacterial cells, followed by limitation digestion utilizing two digestion enzymes, to cut the designated fragment that should including the Deoxyribonucleic acid insert ( if the cloning was success ) , and eventually analysed the consequences utilizing gel cataphoresis.

The consequence showed that civilization A had the recombinant DNA insert ; while civilization B was non, as compared to the positive control included.

As in existent state of affairs, there will be no 100 per centum successes in cloning, due to self-ligation that may take topographic point in the plasmid vector. This sort of experiment is valuable and often employed in Molecular Science to pick out the successfully cloned bacterial settlements, and harvest them for subsequent uses. Furthermore, the combination of limitation digestion and gel cataphoresis, are of import techniques used in limitation function.

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Introduction:

Two bacterial civilizations ( A and B ) were provided in this experiment, the aim was to happen out which civilization the plasmid DNA was incorporating the inserted recombinant DNA fragment ( Sau3A [ 1 ] ) , and besides the size of the recombinant DNA fragment.

In the first portion of the experiment, a commercial kit QIAprep Spin Miniprep Kit [ 2 ] was used to obtain a high quality DNA for subsequent digestion. It involved the readying and glade of the bacterial lysate, the surface assimilation of DNA onto the QIAprep membrane, and the lavation and elution of the plasmid DNA.

The 2nd portion involved the individual or dual digestion of the plasmid DNA, utilizing the limitation enzymes XbaI [ 3 ] and XhoI [ 4 ] . The limitation enzymes recognized peculiar sequences in the plasmid DNA, pBluescript II KS [ 5 ] , to execute the film editing. Therefore, in the dual digestion utilizing both enzymes, a little fragment incorporating the recombinant DNA should be yielded, if the plasmid DNA is positively cloned with the recombinant DNA. This little fragment could be screen out in subsequent gel cataphoresis in portion three of the experiment.

By using an electric field to the agarose gel, the negative charge DNA molecules will migrate toward the positive charge anode. The smaller molecules, the faster or farther they will migrate. Therefore, DNA fragments of different size can be separated. [ ]

Finally, the consequence of the gel cataphoresis could be visualized under UV, as the agarose gel contained the Ethidium bromide ( EtBr ) , which will intercalate with the Deoxyribonucleic acid bases and fluoresce under UV, and the sets become seeable.

Methods:

We followed the process download from WebCT, and we found that there were some different in the portion two and three of the experiment, comparing to the process distributed on the twenty-four hours of practical, and they were stated below.

In portion one of the experiment, since the solution of civilization A was rather clear, in order to increase the output of Deoxyribonucleic acid from it, we typically pelleted 1.5 milliliter twice in the same tubing, therefore obtaining a pellet from 3 ml civilization.

We followed the processs in the practical manual to lysed the bacterial cells, precipitate the genomic DNA, so applied the supernatant to the QIAprep column, to let the plasmid DNA trap in the membrane by affinity. Then washed the column to take hint nuclease activity and drosss, and eventually we get the high quality purified DNA elutes from the column.

Then we proceeded to portion two, the limitation digestion. Six tubings were setup with different digestion enzymes added for each civilization as tabulated below ( other ingredients and inside informations can be referred to the practical manual ) :

1

2

3

4

5

6

Deoxyribonucleic acid from Culture Angstrom

Deoxyribonucleic acid from Culture B

XbaI

XhoI

XbaI and XhoI

XbaI

XhoI

XbaI and XhoI

N.B. The sum of 10x BSA used in each tubing was 1?l alternatively of 2?l.

Then we proceeded to portion three, the Gel Electrophoresis. The gel was setup as follows:

Well 1-10 assignment:

1kb Deoxyribonucleic acid ladder

Culture A ( good 2-5 )

Uncut Deoxyribonucleic acid

Deoxyribonucleic acid cut with XbaI

Deoxyribonucleic acid cut with XhoI

Deoxyribonucleic acid cut with XbaI and XhoI

Culture B ( good 6-9 )

Uncut Deoxyribonucleic acid

Deoxyribonucleic acid cut with XbaI

Deoxyribonucleic acid cut with XhoI

Deoxyribonucleic acid cut with XbaI and XhoI

Control DNA with the Sau3A fragment

Culture A

Culture B

Anode ( + )

1 2 3 4 5 6 7 8 9 10

Wells

Direction of DNA running

Cathode ( – )

Figure 1. Conventional drawing of the place and assignment of the well, and way of DNA migration.

N.B. 2?l of lading dye alternatively of 4?l was added to each DNA solution before lading to the Wellss. And 10?l of samples and marker were loaded into the Wellss alternatively of 3?l.

When the cataphoresis was completed, the gel was put into the Gel Imaging System [ 6 ] to acquire the digital images of the gel under UV, and copied to our USB device.

Consequences:

Figure. 2 below, shows the original exposure taken from the gel imaging system ( on the right ) and a diagram from the practical manual demoing the 1kb DNA ladder graduated table for comparing ( on the left ) .

Figure. 2

Figure. 3 below, shows the exposure after seting the gamma and brightness to give better readability at the mark bands. Some of the sets in interested are labelled.

Recombinant DNA fragment in Culture A

506,517 bp

1018 bp

3054 bp

Vector DNA fragment in Control

Recombinant DNA fragment in Control

4072 bp

By comparing to the control in lane 10, which contains the recombinant DNA fragment ( the lower set ) , we can detect that merely in lane 5 ( dual digestion of civilization A ) had a set at similar degree, although it is weak, but was still rather clear. We did non detect any set at similar degree in the lanes of civilization B.

The consequence showed that civilization A had the recombinant DNA insert ; while civilization B did non.

By comparing the 1kb DNA ladder, the mark fragment in lane 5 ( civilization A ) is about 600 base brace, by unsmooth relative computation. A more accurate consequence can be done by plotting a graph of log size ( bp ) of the DNA fragment against the distance migrated ( centimeter ) [ 7 ]

However, in our instance, the sets of the DNA ladder were non cleared and distinct plenty to execute more accurate measuring.

Therefore, we can reason that Culture A contained the mark recombinant DNA fragment, Sau3A, which was about 600 base brace ; while Culture B did non.

Discussion:

In lane 5, was the dual digested Deoxyribonucleic acid from civilization A, which should incorporate a big and a little fragment. The little 1 was the cut fragment with the recombinant DNA insert, the Sau3A, the size should be around 700 bp, and our consequence was about 600 bp, closely matched with the theoretical value. While the big fragment was the staying vector part, the size should be about 3kb, and our consequence was corresponded to the place of 3054 bp.

Lane 3 and 4, were the individual digested Deoxyribonucleic acid from civilization A, which should be additive molecules after cutting. So, the sets theoretically should be around 3700 bp, which was the amount of the size of the recombinant DNA insert and the plasmid vector. What we got in the consequence, was about 4000 bp, corresponded to the DNA ladder. It was besides closed to the theoretical value.

Similarly, in lane 7 and 8, which were individual digested Deoxyribonucleic acid from civilization B. Since civilization B did non possess the recombinant DNA interpolation, the fragments size after digestion should be about 3 kitchen polices ( the size of the vector ) , and our consequence was closely matched.

From observation, the separation of sets above 3kb of the DNA marker was hapless, it was hard to hold accurate measuring of the sets in that country. Besides, we could detect that those sets in civilization B were excessively bright in fluorescence doing the sets excessively thick. This might be due to excessively much DNA loaded to the gel, or the cataphoresis was non long plenty, that caused the sets to bunch up.

We would propose to reiterate the experiment with less sum of DNA loaded and longer cataphoresis to do the sets separate better and clearer.

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