Dna Isolation Lab Report

PLASMID DNA ISOLATION, RESTRICTION ENZUME DISGESTION AND AGAROSE GEL ELECTRIPHORESIS
Abstract:
The gel is covered with an ion- containing buffer, such as (TAE), that controls the pH of the system and conducts electricity. overall DNA concentration was lower than expected. Using agarose gel electrophoresis is to separate and visualize the DNA fragment, which is produced by restriction enzymes .

Introduction:
The purpose of this experiment is to measure the size of the fragments of DNA and separate them after using the restriction enzyme. Dr. Jane Yeadon discovers the two kinds of plasmids that we used in DNA to GENOME laboratory, which are PJY5 and PJY48. She study two different fungal strains one of them is weak and another is strong, the
one with weak recombination is called StLawrence , and the second with strong recombination is called Lindegren. Moreover, she created two kinds of plasmid PJY5and PJY48 by injected them into bacteria. That gives the plasmids different sizes and sits for restriction enzymes. The purpose of Gel electrophoresis is to separates macromolecules according to their size and charge, which is a basic biotechnology technique. This kind of technique used to analyse and manipulate samples of DNA & RNA or proteins. In this laboratory we would use the Agarose gel electrophoresis to separate and characterize dye molecules of various sizes and charges. The Agarose gel acts as a matrix that slows down the segments as they move to the opposite charged end of the gel. A larger segment will have a tougher time moving through the gel, while a smaller segment will move faster because it is easier to move it through the gel. Agarose gel is a porous gel that allows pieces of DNA to pass through it. In this experiment we used 1X TAE buffer which is a physiological pH because the TAE buffer helping for visualization of really small fragments.
Aims:
The aim of the experiment was to identify the unknown culture A and B as carrying either the pJY5 or pJY48 plasmid. To prepare the media of the two E.coli HB101 that contains PJY48 plasmid and the other one is PJY5 plasmids; and the most important aim is to isolate the DNA plasmid from the culture of E.coli and start to incubate them.
Using the Bam H1 and Hpa1which called the restriction enzymes that to help for digesting the plasmid DNA isolation.
Using agarose gel electrophoresis is to separate and visualize the DNA fragment, which is produced by restriction enzymes .
Method:
The technical staff in the laboratory already prepare the culture of E.coli to the students, which they received two different kinds of E.coli, one carry PJY5 plasmid and the second carry the PJY48 plasmid, we as students should determine between culture E.coli strain HB101 (PJY5) and E.coli HB101(PJY48). By using the Wizard plus SV Minipreps DNA purification system we can isolate the plasmid DNA from the E.coli cultures. To digesting the plasmid of DNA we were using the restriction enzymes (Bam H1) and (Hpa1).
Table 1: The preparation of restriction enzyme samples: BUFFER DNA H2O ENZYME
A.undigested ------ 10μl 40μl ------
A.BamH1 5μl 10μl 34μl 1μl
A.Hpa1 5μl 10μl 34μl 1μl
B.undigested ------- 10μl 40μl ------
B.BamH1 5μl 10μl 34μl 1μl
B.Hpa1 5μl 10μl 34μl 1μl

Incubate the restriction enzyme digests overnight.
By using 0.8% of Agarose gel electrophoresis that about 30 ml to sit the wells. Carefully remove the comb from the gel without making bubbles that will gives a clear picture in the end.

Table 2 : the samples preparation:
Marker 1 2 3 4 5 6
2μl of DNA ladder + 10μl of water 2μl of
Dye+ 10μl of A 2μl of Dye+ 10μl of BamH1
A 2μl of Dye+ 10μl of Hpa1
A 2μl of Dye+ 10μl of B 2μl of Dye+ 10μl of BamH1
B 2μl of Dye+ 10μl of Hpa1
B

The purpose of adding Dye is to make DNA visible and enable to sink to the bottom of slots. The DNA samples + stain are loaded into the wells at one end of the gel. A power supply is added, where the positive electrode is at the opposite end to where the DNA has been loaded. Since DNA is negatively charged, it will move through the gel toward the positive end. Smaller fragments move easier and quicker, while larger fragments move slower.
When the power is shut off, the gel is photographed with a special UV camera that recognizes the dye. The smaller fragments will be at the bottom (faster moving), and the bigger on the top (slower moving)
Results: Figure1:
This figure shows the image of gel electrophoresis that illustrate the plasmid A&B
Result 2:
Table (2): shows the result of DNA concentration in sample A&B
SAMPLE 260 280 NG/UL
A 0.628 0.294 20.7
B 1.046 0.508 34.5
A= 20.7 ng/ul (nano drop) ----- 100 ul of DNA
Loading 10 ul of DNA to the wells , which gives us the concentration of DNA for the wells A = 10*20.7= 207ng
B= 34.5 ng/ul (nano drop) ----- 100 ul of DNA
Loading 10 ul of DNA to the well, which gives us the concentration of DNA for the wells B : 10 * 34.5= 345
DISSCATION:
The first lane that contains the marker was fine.
We are able to measure the size of each band based on bp. It can be seen that, there are difference between lines A and B, the bands in B shows more light then the bands in A, therefore the concentration of DNA of B is higher than A. As a result, the intensity of B’s bands is greater than A’s bands. In line 2 the plasmid DNA is uncut which is expected while the line 3 A with Hpa1 plasmid DNA was uncut which is unexpected. This is due to different reasons such as the quality of enzyme was not good enough to cut the plasmid DNA, inaccuracies in the Elution step, the measurement of the restriction enzyme was not accurate, or there was any kind of contamination. Line4 has 2 bands one at the size of 6557 bp and the other at approximately 900bp which is lower and not that much clear. The band in line 5 Bam1 was 6800 bp which is expected and confirms our result that A is PJY5. In line 6 there is two bands on top of each other one is about 4361 bp and the other 4000 bp which means that both together is 8361 bp. This confirms our result that B is PJY5 (8361 bp). There are 3 types of DNA for our control:
super coiling, naked and Lenya. In line 7 we can see those three types starting from the bottom there is the supercoiling one and above that there is the lenya one followed by the naked. Overall, each size of each band has successfully told us that the A is PJY84 and B is PJY5. Figure 2: the maps of plasmids, it is shown the “cut” sites in (bp) and the location of restriction enzymes.

Conclusion:
In this experiment, by using Mini-prep kit system we had successfully isolated the plasmid DNA. Then, it used the restriction enzyme to digest the plasmid DNA. Gel electrophoresis and 1X TAE buffer helps us to determine which A plasmid and B plasmid. In other word, which one is PJY84 and which one is PJY5. Moreover, we can easily found out that A is PJY48 and B is pJY5 Based on the size of each band in each line.

Reference: