PUC19 Derivatives and Cloning Vectors

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The experiment will primarily involve carrying out a mutagenesis procedure. There will be need of making use of particular derivatives to ensure that the experiment becomes a success. Hence, the plasmid cloning vector pUC19 derivatives will be used (Weyman et al, 2015). The genes contained within the pUC19 are the most targeted part of the experiment. Their property of being ampicillin resistance makes them more preferred to be used in carrying out the whole experiment; the main reason is that they become the important marker for any transformation that is expected in to happen through the experiment. The lacZ gene is also contained in the plasmid vector making it more preferred in carrying out the management procedure. The primary target is always to identify the recombinants in the clone in the reaction (Karpinski et al, 2016). The focus becomes a success with the polylinker inactivating the lacZ gene. The result will be the production of the colorless colonies on the X-gas plates. The production serves as a significant distinction from the non-recombinant clones that result in the production of blue non-recombinant clones.

The derivative pUC19 has diverse variants. In the present case, there will be a need for making use of pUC19M (Cotter, Hill and Field, 2015). One of the common properties associated with such a plasmid is that it has the mutant lacZ gene hence; it will be colorless in the X-gal plate. In the mutagenesis primer, there will be a need for the introduction of a single base substitution. Such kind of reaction will lead to the replacement of the stop codon by the trp codon. The replacement has a significant effect of leading the complete translation of the lacZ gene. Hence, the site directed mutagenesis would target to facilitate the reversion of the mutation that is experienced in pUC19M to wild-type (Maisnier-Patin and Roth, 2015). Reversion will result in the production of different results in the present case being the production of blue colonies on the X-gal plate. The result will make it easy to determine the efficiency of the mutagenesis without undergoing any form of sequencing. The lacZ gene acts as the dominant mutant in the pUC19M, a property that is brought about after the introduction of the stop codon in the reaction.

21 base long premiers will be selected; it will later be annealed to a sequence having the Ndel site in pUC19 as its surrounding at position 183. CATATG is the recognition sequence contained in the Ndel (Yamada et al, 2015). However, the selected premier has set designs that help in the facilitation of change of sequence contained in the Ndel back to CAAATG. The result will be the removal of the site to generate molecules that will not be easily changed by Ndel.

M21 Primer and Mutagenesis Procedure

There is the providence of pUC19M DNA that has been taken through the denaturation process. The denaturation process involves the heating of the derivative through a temperature of 100 degrees Celsius for approximately 3 minutes.

14 μl pUC19M DNA that has been denaturized

In addition to that add annealing buffer of approximately 2 μl

The mutagenesis primer should also be added amounting to 2 μl

There will be the need to also add 2 μl of selection primer

1. Ensure proper heating of the sample at a temperature of 60 °C for 5 minutes. After the heating coming to an end, chill the sample on ice.
2. The liquid will then be collected after properly spinning the sample
3. To the collected sample of 20 μl ensure to add:

Synthesis buffer amounting to 3 μl

T4 DNA polymerase of approximately 1 μl

T4 DNA ligase amounting to 1 μl

Water amounting to 5 μl

1. Ensure to properly mix the sample. Thereafter spin iyt briefly and put it in an incubator having a temperature if 37 °C. The incubation process will have to last for 2 hours.
2. After performing the whole procedure, there will be need for labeling the sample. Place it in the students sample box located on the front desk. You will thereafter be needed to add your details on the record sheet on the corresponding box.

1. Make use of the sterile water in carrying out the dilution of the 2 μl reaction mixture 5 fold.
2. Properly add 1 μl of the DNA that has undergone dilution to competent cells of E. coli BMH 71-18 mutS amounting to 50 μl. After the addition ensure that incubation of the mixture is done for about 15 minutes.
3. At a temperature of 42 °C, properly heat the shock cells for about 1 minute. After the heating, ensure to add the L – broth amounting to 950 μl.
4. Carry out an incubation of the mixture for about 25 minutes in addition to shaking the mixture at 37 °C.
5. Add more L-broth amounting to 4 ml to the incubated sample. Apart from that, also ensure to add ampicillin to the final concentration of approximately 50 μg/ml to a tube with the label ampicillin safety cables. There will be need to allow continuing shaking overnight at a temperature of 37 °C. Hence, you will be need to put it on a rack out the front.

1. Pellet the overnight culture ranging from 1-10 ml through a process of centrifugation for approximately 5 minutes. Pour off the supernatant after the completion of the procedure.
2. Immediately after that, ensure to thoroughly resuspend the pellet containing the cell resuspension solution of approximately 250 μl.
3. The resuspended pellet will have to be transferred to a fresh and properly labeled microfuge table.
4. To each sample, properly add Cell Lysis Solution amounting to 250 μl. Invert the mixture about four times to allow for proper mixing to take place.
5. Add the alkaline protease solution approximately 10 μl. The mixing process will have to be done once more again through the constant inversion. Allow for incubation to take place for about 5 minutes at a room temperature.
6. Add the Neutralization solution amounting to 350 μl. After the addition invert the mixture about 4 times to ensure that the mixture undergoes proper mixing.
7. Centrifuge will later be done at a top speed for about 10 minutes. The process will have to be done at room temperature.

1. By making use of a collection tube, properly insert the Spin Column. Properly label the collection tube after carrying out the insertion.
2. There will be need to carry out proper decantation of the cleared lysate into the Spin Column.
3. Centrifuging will thereafter, be needed to be done at a top speed of approximately 1 minute. The process will be done at a room temperature. Thereafter, the flow through will be discarded and carry out the reinsertion of the column into the collection tube.

1. The wash solution, ethanol, will be used through the process. Add 750 μl of the solution to the mixture and carry out a top centrifuge process that should last for approximately 1 minute.
2. The discarding of the flow through will also be needed and the reinsertion of the column into the collection tube be done thereafter.
3. Properly add the wash solution containing the ethanol to the mixture. The solution should be amounting to 250 μl. Centrifuge should thereafter be done at a top speed of 1 minute.
4. The flow through after that will undergo the discarding process and the reinsertion of the column be done on the collection tube.
5. There will be need for the membrane to undergo thorough drying. This will be achieved by centrifuging the mixture at a top speed of 2 minutes.

1. To the 1.5 ml sterile microcentrifuge tube, cautiously transfer the spin column to it. Care must be highly observed during the transmission process ensuring that none of the column wash solution is transferred to the Spin Column.
2. Add Nuclease Free Water amounting to 100 μl to the spin column.
3. Carry out a centrifuging process at a top speed of 1 minute.
4. Properly label the mixture and place it on ice.

There will be need to create a digest making use of the plasmid DNA pool. The DNA pool should be accounting to approximately 10 μl. Ndel should be present during the digestion process making use of a total volume of 20 μl. Thereafter, there will be need to carry out an incubation for approximately 2 hours maintaining a temperature of 37 °C. The reaction will involve a mixture of the following DNA drawn from the plasmid prep amounting to 10 μl. Sterile water approximately 7 μl will also be needed. Furthermore there will be the 10x Ndel buffer anointing to 2 μl and Ndel of 1 μl.

1. Making use of a sterile centrifuge, spin the 10 ml late log phase JM101 cells at a speed of 5000 rpm. The spinning should last for 10 minutes.
2. To recover the cells in 4 ml of 100mM CaCl₂, there will be need to tip off the supernatant. Thereafter, there will be need to resuspend by mixing and then respining at a speed of 2500 rpm for 10 minutes.
3. Making use of 100 μl of CaCl₂, recover the cells and thereafter mix them properly.
4. Place it on the ice for approximately 20 minutes.
5. Recover 50 μl into each of the two pre-chilled labeled microfuge tubes after properly mixing the cell suspension.

To the other tube add the plasmid prep that did not undergo digestion. It should amount to approximately 2 μl.

1. Carry out proper mixing of the stand for about 20 minutes.
2. Carry out the heating of the shock cells at a temperature of 42 °C. The heating should be done for 1 minute after which there will be a need to add L- broth amounting to 950 μl.
3. To facilitate the incubation process, put it in a lidded rack for about 30 minutes at a temperature of 37 °C.
4. Carry out a brief mixing of the samples and spate them into 500 μl amounts. Place them onto nutrient agar plates containing the amphicilin, X-gal and IPTG.
5. Place the mixture on the correct rack being keen on the rack labels allowing them to undergo incubation through the night at a temperature of 37 °C.

The experiments contributed to the identification of the significant components of the proteins and promoters (Packer and Liu, 2015). Every experiment does always have an aim and a target to be attained. The experiment was significantly involved in the understanding of the contents of the genetic processes. One of the common processes was the DNA conformational analysis. Apart from that, at the end of the experiment, there was a significant understanding regarding the different methods that transposition does function in addition to recombination in the site-specific areas.

There are several techniques of carrying the site mutagenesis process. In most occasions, the processes involved using a single-stranded template, especially the M13, was used in carrying out the mutagenesis process. This became a success by annealing a synthetic premier characterized by around 20-25 bases of length, a defined change has always to be incorporated in the premier. Some of the common changes that are expected are the single or the multiple base changes. Apart from that, we have the deletions and insertions. An appropriate polymearase is then used in initiating the second strand synthesis procedure (Vander Horn and Wang, 2017). Through the process, there is the transformation of the different molecules to the particular and suitable hosts. The clones are need recovered and taken through a process of proper checking to determine the appropriate and tight mutagenic change. DNA sequencing plays a major role in facilitating such a role to be a success. On the completion of the identification process of the mutagenic clone, the fragment will be needed through a process of sub cloning into the vector of choice. Such a process will allow for further analysis of the mutagenic clone hence permitting the process of determining the several other properties associated with the sub clone.

Site-Directed Mutagenesis and Selection of Mutated Molecules

One thing that has to be observed through the experiment is the changes that are to be observed during the mutagenic changes. The phenotype stage Eppendorf is not associated with such changes in most cases. Therefore, in dealing with the situation, there is always an increase in the levels of efficiency that allows for the sequencing of the inordinately large number clones needed to facilitate the identification of the change is not made necessary. As a result of the process, the mutagenesis has been mitigated against being done on a double plasmid strand. The main reason is that by doing so, there will be high levels of difficulty experienced in getting not only sufficient but also pure DNA. Apart from that, the completion of the denaturation process is also made difficult, hence, the complete denaturation of the template in inhibited. The result is that binding of the mutagenic primer in interfered with at the end of the day. However, there has been the invention of several techniques that makes it possible for the site-directed mutagenesis to come to a completion on almost any it strand template. The process has been termed a success citing its ability of incorporating a method that efficiently generates the selection of the mutated molecules (Li et al, 2015).

The Blue-white screening was one of the efficient screening methods. There was a need for deleting the lacZ gene citing the property of leading to a non-functionable β-galactosidase enzyme, the provision of the DNA encoding in the section of the amino cells allowing for the existence of a functional enzyme (Zhang et al, 2015). As a result, the α-complementation was brought to a completion. The results of the experiment were observed on a plate. The components with a whitish cream color of standard E. Coli serve as a representation of the colonies with an insert having the plasmid. The colonies do have a non-functionable β-galactosidase that form the primary cause of the whitish cream color being on display. On the other hand, intact β-galactosidase facilitates the production of a pigment from x-gal. The pigment is responsible for the turning of the turning of the bacterial colony to becoming blue. Transcription of the lac operon is also an essential process to site-directed through the whole process. The process is made a reality through the inclusion of the IPTG.

Some of the precautions to be observed through the experiment are that there is a need for making use of a good control through the whole process. It will ensure the proper transformation of the plasmid without making use of the insert. The result is that all the colonies contained in the particular plate are to be blue (Molinski et al, 2015). The result will serve as an indication that there is proper working being on display by the IPTG and x-gal. The process should also be done without any form of a rush being experienced. The time will allow enough time for the proper expression of the intact β-galactosidase. In addition to that, it will too allow enough time that will facilitate the processing of the x-gal to being a blue pigment.

References

Cotter, P., Hill, C. and Field, D., University College Cork, 2015. Nisin derivatives and the use thereof. U.S. Patent 8,987,193.

Karpinski, J., Hauber, I., Chemnitz, J., Schäfer, C., Paszkowski-Rogacz, M., Chakraborty, D., Beschorner, economics., Hofmann-Sieber, H., Lange, U.C., Grundhoff, A. and Hackmann, K., 2016. Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity. Nature biotechnology, 34(4), p.401.

Li, P., Li, J., Guo, Z., Tang, W., Han, J., Meng, X., Hao, T., Zhu, Y., Zhang, L. and Chen, Y., 2015. An efficient blue-white screening based gene inactivation system for Streptomyces. Applied microbiology and biotechnology, 99(4), pp.1923-1933.

Maisnier-Patin, S. and Roth, J.R., 2015. The origin of mutants under selection: how natural selection mimics mutagenesis (adaptive mutation). Cold Spring Harbor perspectives in biology, 7(7), p.a018176.

Molinski, S.V., Ahmadi, S., Hung, M. and Bear, C.E., 2015. Facilitating structure-function studies of CFTR modulator sites with efficiencies in mutagenesis and functional screening. Journal of biomolecular screening, 20(10), pp.1204-1217.

Packer, M.S. and Liu, D.R., 2015. Methods for the directed evolution of proteins. Nature Reviews Genetics, 16(7), p.379.

Vander Horn, P.B. and Wang, Y., Bio-Rad Laboratories Inc, 2017. Compositions with polymerase activity. U.S. Patent 9,688,969.

Weyman, P.D., Beeri, K., Lefebvre, S.C., Rivera, J., McCarthy, J.K., Heuberger, A.L., Peers, G., Allen, A.E. and Dupont, C.L., 2015. Inactivation of Phaeodactylum tricornutum urease gene using transcription activator?like effector nuclease?based targeted mutagenesis. Plant biotechnology journal, 13(4), pp.460-470.

Yamada, K., Aiba, K., Kitaura, Y., Kondo, Y., Nomura, N., Nakamura, Y., Fukushi, D., Murayama, K., Shimomura, Y., Pitt, J. and Yamaguchi, S., 2015. Clinical, biochemical and metabolic characterisation of a mild form of human short-chain enoyl-CoA hydratase deficiency: significance of increased N-acetyl-S-(2-carboxypropyl) cysteine excretion. Journal of medical genetics, 52(10), pp.691-698.

Zhang, L., Yang, Z., Sefah, K., Bradley, K.M., Hoshika, S., Kim, M.J., Kim, H.J., Zhu, G., Jime?nez, E., Cansiz, S. and Teng, I.T., 2015. Evolution of functional six-nucleotide DNA. Journal of the American Chemical Society, 137(21), pp.6734-6737.