Biology Lab Handbook Assignment Sample

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Introduction

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The analysis of the DNA is required for the evaluation of the DNA library. The importance of the DNA library formation is effective for the cloning process also. Due to the effective nature of the cloning process, some of the analysis steps of the DNA are important. In the analysis of the DNA, the extraction of the DNA is important. At the analysis steps of the DNA, pure DNA is important otherwise the analysis can be effective for the formation of the DNA library. The extraction of DNA is the first process for the formation of the DNA library. After the extraction of the DNA, the purity of the DNA is also important and for this, the UV spectroscopic analysis of the extracted DNA is required. After the purity checking the restriction digestion is part of the DNA analysis. In this part, the restriction enzymes are required to be added as per the requirements of the sequence of the DNA. By the restriction digestion the cutting of the DNA can be possible and after that, the ligation of the DNA with the vector and the transformation of the vector within the organism is required or the amplification of the DNA (Esfandani-Bozchaloyi et al. 2019). DNA sequencing is also important and can be possible with the help of the PCR technique of the DNA sequence. The amplification of the DNA is required for the PCR technique. The contamination of the genomic DNA and proteins can be prevented by the isolation of the small nature of the plasmid DNA from the organism. This process of isolation is known as the miniprep. For the amplification of the lux gene, the PCR reaction can be possible. The sequencing of the plasmid can be possible after the PCR process.

DNA extraction

Bacterial genetic information is minimally ordered in comparison to eukaryotic organisms, and that is population and low and expressed as histone proteins. As a result, encoded by genes DNA isolation is quite straightforward and does not necessitate complicated methods. Within the separation of nucleotides, three main procedures or combinations of them may be used: difference solubility, adsorbed techniques, and ultracentrifugation sedimentation. The method used is determined on the sources of something like the Material be separated as well as the applications (Dopheide et al. 2019). The elimination of peptides is a key goal of genomic DNA isolation, which also is performed owing to having a range of chemical characteristics. Different four steps can be mentioned for performing the DNA extraction. For the extraction and the isolation of the DNA, cell lysis is important. In this lysis, the extraction of the nucleic acids with the protein can be possible. For these steps, the addition of the SDS that is the anionic agent is important for the denaturation of the materials within the cells. The catastrophic agents are important to be used for the adverse effect within the cells. The removal of the protein particles from the cells can also be possible with the help of the addition of catastrophic agents. After that, the enzymatic treatment is required for the degradation of the unwanted components within the cell. For the extraction of the DNA, the contamination of the proteins and RNA particles of the cells are required to be removed and for this, addition of the RNase and Proteinase K can also be added. The chelation of the cell materials can be possible by the addition of the EDTA. By the addition of the alkali in which pH is more than 8 the lysis of the cells can be possible. The precipitation of the nucleic acids by the addition of ethanol or alcohol can be effective and for this, the step is important to be done (Lampignano et al. 2020). In this part, the DNA is extracted from the bacterium, Vibrio fischeri. After the extraction of the DNA, the parity checking of the DNA is important and the removal of the contamination protein and RNA is also required. This extraction is important for the preparation of the DNA library. From the extraction of the DNA from Vibrio fischeri the bioluminescence gene can also be obtained that can be present within lux operon.

Overnight culture of the organism, Vibrio fischeri was kept within a dark room for evaluation of the bioluminescent. 100 ml of culture was transferred and centrifuged at 6000 rpm for 10 minutes at 4°C. The slurry of the cells added in the vortex mixture and the breakage of the cell pallet can be possible. The addition of the TES buffer is required and in this way, the degradation of the nucleic acids can be prevented. The maintenance of the stable nature of the pH can also be possible by the addition of this buffer. The lysozyme was added to the cell mixture and was suspended for 15 minutes. After that, the addition of the SDS and EDTA can be possible. The EDTA can aid in the minimization of the nuclease activity. The appearance of the cell suspension can be changed by the gentle movement of the cell culture due to the denaturation of the proteins and other parts of the cell and the vicious appearance can be possible (Xavier et al. 2021). The addition of the phenol was done for the separation of the protein particles from DNA and the proteins can move as the flocculent materials within the aqueous phase of the cell culture after centrifugation. The phenol-containing solution was removed and by this, the reduction of the shear rate of the DNA of the cells can be possible. After that, the DNA contained solution can be placed within the ice. At the time of the precipitation step, isoamyl alcohol can be added as the defoaming agent for the prevention of the formation of the foam. After several times of inversion of the DNA containing fluid, the gentle press of the DNA can be possible with the help of the glass rod. The excess amount of the liquid can be removed to yield a better yield of chromosomal DNA.

The Spectrophotometric analysis of the DNA can be possible by the measurement of the Absorption reading in the UV spectroscopy. The absorption can be measured at 260 and 180 nanometers. And by this, the ratio of the value can be measured and it is important for checking the purity of the isolated DNA. The percentage of the G and C contained can also be changed within the extracted DNA (Majaneva et al. 2018). The absorption in 234 nm divided by the value of 280 nm is higher than 0.5 and this value is 1.61 for genomic DNA of bacteria. From this result, it can be stated that the contamination of the phenol due to the treatment of the phenol can be present within the solution of the extracted DNA. From the plasmid DNA result the ratio of OD₂₃₄/OD₂₈₀ = 0.82. From this, it can be stated that the phenol contamination is lower than the first one. The extracted lambda ladder DNA can be stated here. OD₂₆₀/OD₂₈₀ ratio can also be effective for the assessment of the purity of the DNA. The time of the loading of the DNA within the agarose gel the addition of the gel loading buffer with DNA solution is important.

Restriction digestion

This restriction digestion can also be known as restriction endonuclease. And in this process, the cutting of the DNA can be possible and this cutting can be effective at specific sites. In this process, some of the restriction endonuclease enzymes are important for the action of the cutting at the specific site of the DNA (Puria, and Nain, 2019). The specific temperature is 37°C and this temperature can be fluctuated to stop the digestion process. The addition of the EDTA can also be effective for the prevention of the digestion process. The heat inactivation is effective also. SalI was used to digest a small product of Vibrio serious and potentially fatal bacterial Chromosome that had previously been extracted, refined, and measured. Following cDNA plus pGEM plasmid digestion, ligase reactions having various ratios comprising 1:1, 2:1, 3:1, and 4:1 have been created. The number of chromosomal DNA insertions accessible for confluence varied amongst the four associated with impaired. The specimens were separated by electrophoresis over an agarose matrix and examined under UV irradiation that use the fluorescence characteristics of EtBr to evaluate the effectiveness of either the restriction endonucleases and also the binding processes (Karthika et al. 2021). The fragmentation lengths for such bands visible on the solutions were calculated using λ ladder's calibration curves.

For the cloning, the restriction enzymes are required to be selected and for this experiment the selected plasmid is pGEM and the ampicillin resistance gene is also required to be inserted within the plasmid. The ligation of the lacZ gene is also important for alpha complementation. The protocol is possible through agarose gel electrophoresis. After the extraction of the DNA, the cutting of the restriction endonuclease can be possible by the addition of the restriction enzyme with the DNA solution within a vial. SalI is the main restriction endonuclease that is specific for cutting the chromosomal DNA of the bacterium. After that, the vector can be ligated and the construction of the DNA library can be possible. For the preparation of the agarose gel, the agarose powder was mixed with the 1X TAE buffer (Ekawasti et al. 2022). The volume of the gel can be changed according to the size of the gel. The suspension was heated and then it remained to solidify. After that, it was cooled. At the time of the preparation of the agarose gel, the addition of 1 microliter of EtBr is required. 20 to 30 minutes was required for the solidification of the gel. The comb was also placed before remaining for solidification of the creation of the well within the gel (Nagar et al. 2020). The addition of the EtBr is also important for the visualization of the DNA band within the agarose gel. The final concentration of EtBr is 10 millimolar. The chelation of the magnesium ion can be possible by the addition of EtBr. In the different tubes, the control and the examined DNA can be retained. The control can be prepared by the addition of the restriction buffer with chromosomal DNA. Within this, no enzyme is not present here. Another control mixture can be prepared with the chromosomal DNA and TE buffer. After the process of restriction digestion, the results can be mentioned below.

From the result, the size of the DNA can be possible by the comparison of the size of the DNA from the ladder DNA. Restriction digestion is effective for the evaluation of the size of the DNA bands that are extracted from the bacteria (Boers et al. 2018). The DNA band can move according to the small and large size of the DNA through the agarose gel. By the comparison from the ladder DNA fragments it can be stated that the in the first line the fragment is contained with more than 21226 base pairs. Then in next three column of the gel the DNA is high from 21226 bp. In the fifth segment the DNA is composed of about 300 base pairs. Then the DNA composed of about 2021 base pairs. Ladder DNA is loaded at the first and last well of the gel. From this it can also be stated that in the sample the RNA contaminations is high or the amount of the plasmid DNA is also high and for this the rate of movement of the supercoiled DNA is high within the sample of the DNA.

Ligation and transformation

In the DNA cloning process, ligation is an effective process. After the cutting of the DNA fragment with the help of the restriction endonuclease the addition of the DNA fragment within the vector is important. For this process, ligation can be possible with the help of the ligase enzyme. For the ligation process maintenance of the specific temperature that is the room temperature is important. The function of the enzyme, ligase, is possible at room temperature. Phosphatase treatment for this process is required for the prevention of the recirculation of the vector (Liu, and Li, 2018). The mixture for the ligation process includes a cut vector by salI, TSAP enzyme, and water.

Explanation of results and table

The mixture of cut vectors by salI of 9 microliters, TSAP enzyme of 1 microliter for the phosphatase treatment, and in which the water was not added. Another mixture was prepared by the addition of cut vectors by salI of 9 microliters and 1 microliter of water and the total volume for both cases is 10 microliters. The mixture was incubated for 15 minutes and the heat inactivation can be applied for the prevention of the action of the TSAP enzyme. The size of the fragments of the lux gene is 9 kb. The size of the vector is 3 kb and the concentration of DNA can be measured at micrograms (Conibear et al. 2018). The number of the base pairs can be six. The micro-centrifuge tubes can be leveled with P0, P1, P2, P3, and P4 in case of the phosphatase treatment and for the untreated sample U0, U1, U2, U3, and U4 can be mentioned. The amount of the genomic digest can vary and the amount of the genomic digest can be 0 microliters, 1.5 microliters, 3 microliters, 4.5 microliters, 6.0 microliters can be added and the volume of the added water can also vary. The amount of the 2X buffer is 10 microliters for each of the Eppendorf. The amount of the genomic DNA is the same in the case of the sample which is treated with phosphatase. For the lambda DNA, the T4 DNA ligase is important for the ligation process. 12 tubes are used and from this, tube 1 is the control in which any of the DNA samples was added. In tube 2 the positive control is formed by the addition of the plasmid DNA and vector DNA. The formation of the competent cells is important for the ligation of the gene and the transformation of the vector within the organism. For transforming the vector within the organism the process of the transformation is required (Dunkelmann et al. 2018). The ability to take up the gene containing a vector by the bacterium can be possible by the transformed bacteria. This transformation can be possible with the help of the treatment of calcium chloride. The pores within the membrane of the bacteria are required to be sufficient for the transformation of the foreign DNA and this heat shock is required to be applied. Blue-white screening is the common experiment for checking the transformed bacteria. In the presence of the IPTG medium the organism can be incubated (Deaner et al. 2018). And then in the media, the X-gal can be added. If the organism is transformed with the ampicillin-containing gene the formation of the blue-colored colony can be possible within the Luriaberteni agar media. This phenomenon can be known as the blue-white screening.

Expected results and their improvements

The expected results can be the formation of the blue colony and from this, it can be stated that the transformation process of the organism can be successful and for this, the transformation of the gene to the organism can be effective. The ligation and the transformation process of the DNA within the organism are important for the formation of the genomic library with the recombinant nature of the gene. The improvements in the case of the improper process of the transformation of the vector within the organism process of the transformation such as the incubation time and the process of the heat shock are required to be proper (Jia et al. 2019). The ligase enzyme is required to be added at the last step otherwise the action of the ligase enzyme cannot be effective. The amount of the loaded DNA is 20 microliter within the well for the agarose gel.

In the expected result the fragments of the DNA is moreover equal to the ladder DNA fragments. The result is expected due to the standard size of the DNA fragments matched with the adder DNA fragments. The improvement of the result is important for the development of the resulta) Plasmid miniprep

Restriction digest

For the preparation of the pure DNA library, the isolation of the pure nature of the plasmid DNA is important. For this the isolation of the plasmid DNA is important. As the amount of the plasmid can be lower due to the low amount of DNA within the bacterial cell this process can be referred to as the plasmid-miniprep. For the preparation of the plasmid miniprep the restriction digestion is also important (M Kurdi Al-Dulaimi, and Ariffin, 2019). The miniprep is done in this part for the examination of the bioluminescent. In this process, the purification of the plasmid DNA is also important. Rapid boiling is the important part of this process that is done after the inoculation of the overnight culture is required. After that, for the observation of the bioluminescence, the lux clones containing genes are also required to be inoculated in the culture. For the presence of bioluminescence, the temperature is required to be kept at 28°C. In this miniprep process, the addition of the pGEM and the lux clones containing culture is required to be incorporated. For this purification process, the use of the treatment with RNase and phenol is important for the removal of the RNA and protein contamination from the plasmid DNA (Andy, and Okpo, 2019). the lux operon containing can be isolated from the bacteria, Vibrio fischeri, and then the pUC18 plasmid can be recombinant with these clones, and then it can be transformed to the E.coli DH5-alpha strain for the rapid growth of the lux operon containing the organism.

From the table, it can be stated that the amount of the water can vary and for this, the concentration of the culture containing DNA to uptake the insertion size of the lux operon can also vary.  The total volume after the addition of the buffer, water, restriction enzyme, and the amount of the DNA. In each tube containing the blue-white colony the amount of the DNA and restriction enzyme is the same. In the case of the pGEM plasmid, the amount of the DNA is 1 microliter and in this case, the added amount of the water is 3 microliters for each of the tubes, and for the pGEM plasmid, the taking of the water is 6 microliters (Nguyen et al. 2018). With the help of the restriction enzymes, the lux gene can be cut and then ligated to the plasmid then it can be transferred to the E.coli DH5-Alpha and after the transformation, the recombinant cells can be grown for the product of the gene.

Plasmid + insert size

After the extraction of the plasmid from the cell the application of the heat is effective for the denaturation of the chromosomal DNA due to the presence of the high nature of the shear rate. The plasmid DNA can be intact. The protein can be coagulated and can be removed by the process of precipitation. In the solution after the centrifugation, the plasmid can be present and can be precipitated by the use of ethanol and suspended by the use of the TE buffer. The size of the inserts plasmid can be evaluated with the help of electrophoresis (Shokry et al. 2021). The insertion of the gene size can be possible within the plasmid with the help of the ligation process. The restriction enzyme is important for the cutting of the gen and this type of restriction enzyme can be different that can be sticky end cutter and blunt end cutter. It is the main step of DNA cloning. In the cloning process, the recombinant plasmid can be obtained and for this, the insertion of the new genes within the plasmid can be possible. The size of the gene that is inserted within the plasmid can be said as the insert size. A plasmid is known as the extrachromosomal DNA. For this experiment, the amount of the insertion size is 2 kb. For the insertion of the gene within the plasmid, the polymerase chain reaction step can also be applied such as annealing for joining the inserted portion of the gene within the plasmid.

Electrophoresis

The transformation is required to be proper and for this, the heating block is required to be set at 37°C and the sample of the gene is required to be loaded within the well of the gel. The ladder DNA can also be loaded for the determination of the insert size within the plasmid. The electrophoresis can be possible by the set of the cathode and anode. The TE buffer is also important for the maintenance of the actual pH. Due to the presence of the SDS which is an anionic dye with the DNA the movement of the DNA fragment can be possible from the cathode region to the anode region due to the presence of the repulsion force with the negative charge. In electrophoresis, the movement of the plasmid DNA can be faster due to the small size of the plasmid DNA. According to the molecular weight of the DNA fragment, the movement of the DNA can be possible (BAryshev, 2022). In the case of the presence of RNA contamination, the movement of the RNA can be higher than due to the presence of a single strand of RNA. After the application of the electric current, the DNA movement can be possible and the identification of the inserted gene and its extraction can also be possible.

(b) Polymerase Chain reaction

Lux gene present

By the process of the polymerase chain reaction, the amplification of the DNA can be possible.  For this process, three steps can be mentioned (Ramesh, and Mohanraju, 2021). One of the main three steps is denaturation and in this step, the unwinding of the DNA strand can be possible. For this, a specific temperature is required to be applied in this step. The temperature is 94°C to 96°C for this step. The second step is annealing of the inserted portion and for this, the attachment of the inserted DNA fragments is required that can be cut with the help of the restriction enzyme. In this step the specific temperature is 55°C. This temperature is required for the attachment and also for the unwinding of the sequence from the DNA base (Hamdi et al. 2021). As the rate of attachment can be high so the chances of the error within this process can be high and for this, proofreading is another kind of mechanism that is also present in this step. The last step is the elongation which is the replication of the DNA and these steps are required to be repeated for the amplification of the DNA and by the way, the amount of the DNA can be increased and the increment of the gene product can also be possible. Each of the steps is required to be repeated 15 to 40 times. In this way, the cyclical steps of the polymerase chain reaction can be possible.

For the activation process of lux genes of the lux operons, LuxR is the repressor protein that can bind to the 20-base-pair of the inverted repeat. This can also be referred to as the lux box, which is centered with 42.5 base pairs in the upstream region of the transcriptional start of the lux operon. Same lux box-like components can also be identified within only a few of the LuxR-activated promoters of the bacterium, Vibrio fischeri. By the regulation of the repressor protein the rate of the DNA replication can be changed (Badar et al. 2019). The production of the luminescent protein by the bacteria, Vibrio fischeri can be possible by the regulation of the lux operon. This gene can be effective for the production of the luciferase that is responsible for the production of yellow-colored bioluminescence. The absorbance maxima of the bioluminescence are at 490 nm. The organization of the lux operon is luxCDABE. This can be modulated by the presence of the genes that are responsible for the regulation of the binding of the Flavin and its mechanism can also be regulated. After the insertion of the gene within the plasmid, the polymerase chain reaction product can be evaluated with the help of the electrophoresis the process of the electrophoresis can be mentioned and the obtained result can be mentioned below.

Electrophoresis

The electrophoresis is important for the evaluation of the PCR products determination. After the completion of the PCR process, the product can be analyzed with the help of agarose gel electrophoresis (Lestari et al. 2020). From the comparison with the ladder DNA, the changes of the DNA product can be possible. The Identification of the luciferin-productive gene can also be possible with the help of the electrophoresis technique. Due to the production of the luciferin and its chemical reaction with the oxygen molecule the production of light can be possible. The catalyst luciferase can also be produced for the catalysis of the reaction and this speed up of the reaction can be possible. The addition of the TE buffer and the right percentage of the agarose gel is important for the solidification of the gel.  The solidification is required to be proper due to the movement of the DNA fragments. UV Trans-illuminator is required to be used for the visualization of the DNA bands. The DNA bands that are produced by the PCR process can be analyzed by the BLAST analysis. This analysis can be achieved in the next part and from this analysis, the evaluation of the DNA analysis can be possible, which is the important part of the formation of the DNA library (Green, and Sambrook, 2019). All of the steps, mainly the purification of the DNA, are an important part of the formation of the DNA library that is effective for the availability of the DNA.

4. (c) Sequencing of plasmid

Sequence BLAST research

The BLAST search can be carried out with the help of the NCBI platform and the detailed analysis of the DNA can be possible by this analysis. For this analysis, the sequence of the plasmid that can be the recombinant plasmid containing the lux gene or not can be possible. For this analysis, the sequencing of the plasmid is also important. After the recombination of the gene within the plasmid, the sequence of the plasmid can be assessed (Kopotsa et al. 2019). One of the sequences can be analyzed of the plasmid 500 F (P 500 F). The analyzed result can be mentioned below.

Identification of the gene sequence

The identification of the gene sequence can be possible with the help of the BLAST search of the gene sequence. The result that is obtained by the BLAST analysis can be mentioned in this part and from this, the sequence of the gene can be identified. The identification of the gene is important for the evaluation of the product. With the help of the identification, the presence of the gene within the plasmid sequence can be confirmed.  By the analysis, the gene product can be confirmed (Lv et al. 2020). In the case of the presence of the error within the transformation or the ligation process of the gene, the gene can be missed within the plasmid. Gene sequencing can be possible with the help of the BLAST analysis. The specific gene sequence maintenance is important in the library to get the particular product of the gene.

Conclusion

From the overall analysis, it can be stated that the formation of the DNA library can be possible by the different steps and all of the steps are related to the extraction of the DNA and its analysis. As the construction of the DNA library is important for the analysis of the critical function of the DNA, the purification of the DNA is the important part. In the field of molecular biology, the formation of the DNA library is an important part. For the production of many of the clones of the gene, the polymerase chain reaction is an important part of the amplification of the DNA sequence. DNA libraries can be created by breaking the chromosome of interest into substantial segments with either a restriction of endonucleases, introducing each segment together into vectors, and afterward introducing every vector into the bacterium. To create a genomic collection, each organism's Isolation of DNA the cells but then just processed with both an endonuclease to break the Genome onto particular size segments (He et al. 2019). Applying DNA ligase, individual pieces are set as a framework into a vector. Any plasmid or genomic DNA Fragments library consists of the DNA fragments which thus constitute an organism's comprehensive genomes. A chromosomal collection is formed by first extracting DNA from the cells and afterward amplification of this fragment is required with DNA cloning technologies.