IntroductionFor control reaction and digestion reaction. We started

IntroductionFor this experiment, we are going to digest lambda phage DNA with restriction enzyme, EcoRI endonucleases. It will produce a number of predicted fragments and we are going to identify them. It will produce 6 fragments such as 21226,7421,5804,5643,4878 and 3530. We will be learning how restriction enzymes work and learn about agarose gel electrophoresis.The restriction endonucleases recognize specific DNA nucleotide sequences in double stranded DNA and cut it. The recognition sites will be symmetrical and both DNA strands in the site will have the same sequence reading from 5′ to 3′. They are known as palindromes. Gel electrophoresis is a technique for analysing fragments of DNA after being cut by restriction enzymes.The fragments are separated from each other using gel electrophoresis.ProceduresSetting Up The ControlCollect all equipments required for the experiment. We have to prepare 2 reactions: control reaction and digestion reaction. We started off by preparing the control reaction by transferring all of the contents of the reaction buffer into tube 2 containing DNA using a fresh pipette tip setting the pipette at 40 microlitre.    We then transferred the contents of tube 2 using the same pipette tip to tube 3 setting the pipette at 45 microlitre. Lastly, we labelled tube 3 with my group members initials – now known as control.The transferring of tubes from tube 1 to 2 to 3 is done to minimise the loss of DNA. Setting Up The Digestion ReactionNext, we prepared the digestion reaction by transferring all of the contents of the reaction buffer into tube 2 containing DNA using a fresh pipette tip setting the pipette at 40 microlitre.    We then transferred the contents of tube 2 using the same pipette tip to tube 4 (Eco RI) setting the pipette at 45 microlitre. Subsequently, we labelled tube 4 as reaction with our group members initials. To have a balanced mixture, we tapped both the control and reaction tubes before incubating the tubes for 60 minutes in the water bath at 37 C. After The 30 – 60 Minute IncubationFollowing which, we added 5 microlitre of 10x gel solution to each tube, The purpose of 10X solution is for easy loading and tracking of DNA samples in agarose gel, it also acts as a weighing solution. The samples are ready for electrophoresis after mixing the tubes.  Loading DNA SamplesOnce the water bath has reached the temperature of 65 C, we heated our samples and the standard DNA fragment for 2 minutes. After which, we left our samples to cool for a few minutes while we prepared to place the agarose gel into the electrophoresis and created wells to insert the samples.To ensure that we do not damage the wells or spill the samples, we practiced adding into the wells for 5 times before we loaded the first well with 40 microlitre of the standard DNA fragments and then the reaction and control samples into the subsequent 2 wells. Running The GelOne of my group members prepared the electrode terminals while the others ensured that the samples were loaded properly before  connecting the negative and positive indicators on the cover and apparatus chamber.We ensured that the black wire was inserted into the black input and the red wire is inserted into the red power source.We set the voltage of the electrophoresis to 100 volts and run it till the samples reached to the length of 8cm. To determine that the current is being circulated, we checked for bubbles being formed on the electrodes at an interval of 15 minutes. Since DNA has strong negative charge, it will migrate through the gel towards the positive electrode.Once our tracking dye has migrated to 8cm, we turned off the power and all the other required switches before removing the cover to remove the gel and place it in a tupperware.The gel is then stained by 1x DNA Blue Flash Stain for 5 minutes to ensure that the staining process takes place effectively. The gel has to be fully submerged with the stain of 100ml.One of us prepares a fresh tray with 200ml of water to destain the gel while the others assist removing the excess stain back into the beaker of 1x DNA Blue Flash Stain for other classmates to reuse it.We repeated steps 6 and 7 several times to achieve bands that are visible under UV Light. Lastly, we took photos of the destained gel to meet the requirements and answer the question of the report. We labelled the Standard DNA fragments bands: 23130, 9416, 6557, 4361, 3000, 2322, 2027, 725, 570 bp on the gel and the Lambda DNA bands from Eco RI cleavage: 21226, 7421, 5804, 5643, 4878, 3530 bp on the gel.Results (Picture of the destained gel + Picture of labelled gel) Figure 1 : Destained Gel (Without labelling) Lane 1: Standard DNA samples (S) (from left)Lane 2: Lambda DNA bands cut with EcoRI (tube 4)Lane 3: Lambda DNA (uncut) (tube 3)Figure 2 : Destained Gel (With labelling) Bands we get in the experiment (IN ORANGE)Other Bands (IN RED)Lane 1: Standard DNA samples (S)Lane 2: Lambda DNA bands cut with EcoRI (tube 4)Lane 3: Lambda DNA (uncut) (tube 3)DiscussionIt can be seen that the large fragments can be found at the top of the gel because it ismore difficult for the larger pieces to be strained through the gel. The small fragments will be found at the bottom of the gel. The size of each fragment in lane 2 is estimated when electrophoresed by comparing with a standard ladder of known DNA fragment on lane 1.Based on figure 1, the DNA fragments on lane 1 and 3 are still relatively clear. However, It can been seen that the separation was not done very well on lane 2 as the ladder for the 6 fragments bands did not show up clearly. It might be because the gel was not stained for a sufficient period of time. Based on figure 2, Lambda phage DNA was cleaved with EcoRI, which cuts at 3 different places on the lambda phage shown in our experimental results. However, the expected result that we should get should be 6 fragments bands after Lambda phage DNA is digested with EcoRI. The 6 bands are 21226,7421,5804,5643,4878 and 3530. In order to get the 6 bands, we will need to do several rounds of destaining and staining.Questions1. What are other methods of gel staining?General protein gel stains with Coomassie Brilliant Blue gel being the most frequently used staining gel. They bind tightly to proteins and absorb light in the visible region of the spectrum. As low as 0.1 microgram of protein can be detected using this dye while factors like thickness of the gel and the properties of the protein will also come into play. Colloidal Coomassie can be defined to effectively stain proteins for 1 hour and requires just water (no methanol or corrosive acid) for destaining.Silver staining is the most delicate colorimetric method for identifying total protein. The method includes the discharging of silver onto the surface of a gel at the areas where the protein bands allocated. Silver ions (from silver nitrate in the staining reagent) interact and bind with certain protein functional groups. The strongest connections happen with carboxylic corrosive groups (Asp and Glu), imidazole (His), sulfhydryls (Cys) and amines (Lys). Different sensitizer and enhancer reagents are fundamental for controlling the specificity and proficiency of silver ions binding to proteins and effective development of the bound silver to metallic silver. The development procedure is basically the same with respect to photographic film: reduction of silver ions to metallic silver, bringing about a darker dark shading.General DNA gel stain which is a kind of fluorescent dye. As they have a low fluorescence level, once they bind to the nucleic acid, the resulting complex will become highly fluorescent. Most dyes will result in complexes that can be excited in the ultraviolet range and emit light in the visible region.2. Why do you need to heat the Lamda DNA fragments prior to electrophoresis?The purpose is to heat the DNA of the organism in which you are trying to analyze rather than actually heat up the fragments. Heating of the said DNA allows for the breakage of the double helix which in turn allows them to be split up so that the sequences can be determined by length.3. Predict the number of DNA fragments and their sizes if Lamda phage DNA were incubated and cleaved simultaneously with both Hind III and Eco RI.13 bands (solution below).ConclusionOur experiment was a success as we were managed to view most of the bands after repeated destaining of the gel. We were also able to gain a deeper understanding of restriction enzymes and agarose gel electrophoresis. Restriction enzyme cut at different locations on the DNA because the enzyme has a specific sequence of nucleotides on the DNA that let the enzyme cut on the DNA. The more times the restriction enzyme cuts the DNA, the further it will travel through the gel during electrophoresis. We didn’t able to see all the fragments as bands in the gel. It may be because some bands are so close in size that they did not separate when doing the experiment. Some fragments are so small that they cannot be detected. We could get better results by running the gel for a longer time, using a more sensitive DNA stain or change the gel concentration.References

Author: admin


I'm Mia!

Don't know how to start your paper? Worry no more! Get professional writing assistance from me.

Check it out