Purpose

Purpose

The purpose that will be concluded in labs 9 and 10 experiments is the isolation of chromosomal DNA from Escherichia coli, a bacteria cell, and this extraction will be further analyzed. This is conducted with the use of enzymes and solutions. The analysis is conducted with the aid of Agarose Gel Electrophoresis to help us see the extraction of DNA’s identity and to see its purity. In lab 9 the experiment that was done is preparing the sample and that was done by isolating E.Coli cell and then the DNA was extracted. In lab 10 the experiment was to identify the DNA sample’s identity by the use of DNase and this was conducted under different solutions, enzymes and temperatures. To test the purity of our sample is by the use of Gel Electrophoresis and this shows if there is any RNA contaminating the extraction.

Methods

The steps that were taken in lab 9 was that we started with the suspension in saline-EDTA and the stirring of Eschericia coli. This will allow us to achieve the smooth suspension. The second step was to add saline-EDTA and the following step was to add lysozyme and to incubate this prep for 10 minutes at a temperature of 37C. The next step was the addition of 10% SDS and again incubate this solution at 60C. This step allowed the DNase to be inactivated. A cooling period was later preformed to allow the prep to be lowered to room temperature. The addition of NaCl and chloroform is the following step preformed to our tubes. First we centrifuge and then we extract the aqueous layer that is the top layer. 95% of ethanol was pipetted after. A Pasteur pipet was burnt for a hook to be formed and we can then get the DNA long strand. Concentrations of ethanol were used to wash the DNA strand into. In lab 10, the following lab, after allowing our tube to be frozen in a -20C environment, the DNA after being added to the solution you can follow in the Enzymatic digestion table below, created samples and these samples are analyzed by the use of agarose gel electrophoresis. This part was preformed by carefully pipetting the samples into the lanes.

a) Preparation

As this lab was preformed in two separate labs, in two following weeks, the steps taken were precise with specific amounts and all were for the aim to protect the integrity and to later use nucleic acid’s structure for extraction. The first step taken was to suspend Eschericia coli cell paste in saline-EDTA. Saline-EDTA has the properties that are 0.15M NaCl, 0.1M EDTA and its pH is 8. The second step was to pipet the bacterial suspension ins saline-EDTA (4.5mL) to a conical tube with Blue top tube. This tube contained 0.35g of Eschericia coli. Third, lysozyme (0.25mL) was pipetted which was 20 in our sample. After this step we gently invert to mix the solutions. At a temperature of 37C we incubate this prep. After waiting for 20 minutes we take 10% SDS and add (0.5mL) to the sample and mix to later incubate it at a temperature of 60C. After 10 minutes we allow this prep to reach room temperature. After room temperature was reached we added 1.25mL of 5M NaCl and 6.5mL chloroform to our tubes. After mixing for 10 minutes we centrifuge our prep (at 2500rpm). After 5 minutes of centrifuging we extract the aqueous layer (the top layer). This extraction is obtained by the use of a 1mL plastic pipet. After extraction we placed it in a clean orange top tube. An alcohol layer formed on top of our aqueous layer after adding 95% of ethanol. A partial hook allowed us to extract our DNA. The extracted DNA was submerged in 70%, 85%, 90% and 95% concentrations of ethanol. The STE (0.5mL) was used to dissolve our DNA in. The last step of preparation was to place the DNA in a freezer at the temperature of -20C.

c) Agarose electrophoresis

Agarose electrophoresis was a main component in this experiment due to the fact that it provided the results for us to further examine the nucleic acids after we identify our DNA. The agarose gel provided the seperate DNA data for us to analyze. The way agarose electrophoresis works is that is has a field that is electric and this creates a gradient under the circumstances where the pH is 8 and the amino acid is negatively charged, it then allows nucleic acid’s molecules to go through where DNA’s features is that is has a sugar phosphate backbone and this makes its charge negative. After the bands are formed we can observe the purity of our DNA. To additionally see if our DNA is contaminated or if all the bands are there if not what is it missing, we see those elements by the aid of a picture. The picture shows the DNA and RNA, because without it we can not see them with the naked eye. The DNA and RNA are tiny. The chemical aspects here is a fluorescent compound chemical: Ethidium bromide solution. The ETBR shows the presence of DNA or RNA with the color band.

Discussion and Conclusion

From the legend I arranged and for further observations, could be seen in the picture, DNA only was seen in 5 lanes. These lanes were lanes 1 to 4 and lane 7. This does seem to follow with the steps we preformed, because the samples with DNase did not show clear DNA bands due to the fact that DNase is an enzyme that has the capability to destroy DNA. The breakup I will provide in the following paragraph is from the Results part of this lab report.

In the gel electrophoresis you can see lane 1 has a clear view of DNA and later the rRNA and the tRNA follows. The rRNA and tRNA are closely formed. These nucleic acids form this clearly because their size is small and this condition allows them to go through the gel from below. This result is after the incubation at the temperature of 0C. Also the fact that this prep was without DNase played a role because as I mentioned DNase would have destroyed the sugar phosphate backbone’s phosphodiester DNA bonds.

The lane #2 was a repeat of the conditions of lane #1 which was the incubation at 0C and no DNase added, we see the same results where the DNA is viewed at the top and rRNA, then tRNA.

The third lane there was a new condition that was that there was DNase and here the temperature is now incubated at 37C. Referring to our picture you can see the presence of DNA, so this might be the result of an experimental error or that the timing was off. DNase was supposed to destroy the DNA and it should not be seen. Yet the rRNA and tRNA formed as expected.

Lane #4 had no DNase but was incubated under the same temperature as the sample in lane #3 (37C) and the DNA formed just fine and the rRNA and tRNA following. The reason why the ribosomal RNA and transfer RNA where at the bottom is because of previously mentioned it is small and can pass through quick.

The presence of DNase is also in lane 5 and here it works, because no DNA is shown and DNase did its job. This sample was incubated at 37C.

In lane #6 the conditions were the same as the conditions in lane #5 and you can see that because of no presence of DNA and that is it was affected by the addition of DNase. The rRNA and tRNA showed and this sample was incubated at the temperature of 37C.

The DNA appeared back in lane #7 because in this sample the use of DNA has been switched to tRNA to form the sample. The addition of DNase was in this sample yet it did not seem to have a big effect on the sample because we can see it here.

Both lanes 8 and lanes 9 have the same visual yet both samples were prepared under the different conditions. Where lane 8 does not have the presence of DNase and lane 9 has DNase.

After the analyzing of all the samples and the temperatures they were under and the fact that they had or did not have DNase in them, we can say that none of the DNA was RNase contaminated. The steps taken in these labs showed a conclusion of the use of the agarose gel and for some it did not work. We do see some errors with the job DNase had no preform and it was not always the case that with DNase there is no DNA and even sometime without DNase there would be no DNA. The presence of rRNA and also tRNA (nucleic acids) showed us from the picture that our DNA that we prepared into a sample’s purity was not 100%. Different conditions did play a role in this experiment where there were samples at -20C, 0C, 37C and 60C. This conclusion can definitely be related to some everyday life events where as an example the use of gel electrophoresis is preformed on infants and their potential father to see if he is their biological offspring. Another use that the Biotechnology discovered is that the gel electrophoresis can be used to see the genes that are present in DNA and see if that same gene is in a disease. This is possible through the use of gel electrophoresis because more than one gene can show the this and the lanes in the gel show the clear picture.

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