Need solution urgently What is the correct order to prepare purerecombinant proteins when using E coliexpression system?1. Select appropriate columnchromatography to purify the recombinantproteins2. Make a DNA plasmid containing genethat codes target protein3. Transform the DNA plasmids into E. colicells3. Grow E. coli cells in appropriate media foroverexpressing recombinant proteins byIPTG and harvest cells4. Break open the cells to release therecombinant proteins into appropriatebuffer and spin down to get supernatantpart

Recombinant proteins are proteins that are encoded by recombinant DNA that is cloned in an…

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Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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A amp PBR322 4301 fot B Clear Zones Figure 2 The postgraduate student, Demika, inserted her gene of interest into the plasmid, pBR322, before transformation into the competent host cell using heat shock method. After that she cultured the cells on the Ampicillin agar plate before replica plating the colonies onto another Ampicillin (A) and Tetracycline (B) agar plates shown in Figure 2. (1) Referring to the vector pBR322 in Figure 2, which recognition site was cleaved to insert the gene of interest? Based on the observation above, can you identify which colonies are carrying positive mcombinants of BR322? Explain your selection.
Kha Vu Danels Include: 8DX : Safehy Jor bromie tnto lab repart! Name Section date sheet MAPPING PRACTICE #1 Below is a restriction map for the plasmid PGEN 101 (total length = 20 Kb). Using this map as a guide, give the number of restriction fraqments along with their associated lengths that would result from digesting PGEN 101 with the restriction enzymes EcoRI, BamHI and a combination of ECORI and BamHI. BamHI 3.2 Kb 1.7 Kb EcoRI BamHI PGEN 101 8.7 Kb 5.5 Kb .9 Kb EcoRI ECORI DIGESTION PERFORMED SIZES OF FRAGMENTS OBTAINED 10.4 kb , 0.9kb, 8.7 Kb EcoRI 3.2 Kb, 16. 8kb BamHI EcoRI + BamHI
5 μL of plasmid DNA (pUC 19) was added to 50ul of CaCl2 competent E. coli that was then added 250 μL of SOC medium, 100 μL of this solution was plated onto a TSA + ampicillin plate. Can you please show me how I calculate the total number of successful transformants per mL of competent cells plated. The total number of colnies counted on plate was 70. Thank you
During which step in the transformation protocol does the plasmid DNA move into the cell? Heat Shock Grow in LB media at 37°C Plate on media with Ampicillin O Incubate the cells on ice with CaCl2 in the absence of plasmid. fg f1o 19 %24 & 5 6. 7 8. 4 5 R Y U P F G J 2 K 3] vの

One of the frist steps in isolating plasmid DNA via mini-prep is to pellet the cells after O/N culture and then resuspend them in buffer P1. What's the point of pelleting the cells just to resuspend them again? To concentrate the cells P1 is a buffer, preventing small changes in pH O This step is not absolutely necessary P1 begins the lysis process D Question 2 What would happen if we didn't centrifuge the tube containing lysed cells after adding neutralization buffer and instead just added right to the column? O We could fihish the prep but we would have protein/RNA contaminants at the end O The precipitate is less dense than water so it shouldn't get in the way since it floats on top O The precipitate would obstruct the column and our prep is ruined O Our yield would be reduced, but we'd get something
Help Please Supercoiled DNA migrates through an agorose gel faster than similarly sized linear DNA? True or False? Using the plasmid map, indicate where (bp region on plasmid) these enzymes cut on the plasmid. EcoR1 1. 1 Hindlll 2. 637 Bpu101 3. 14 BamHI 4. 256 To sequence a DNA plasmid, you need 1 µg of DNA. If your DNA concentration was 0.45 µg/µL. How many µl of your DNA 0.45 µg/µL is required to get 1 µg final? Answer in µl using 2 decimal places. Your Answer: Answer units >
3A. Following transformation with the CRISPR plasmid, the yeast will be plated on SD-URA plates. Why is SD-URA media used? Name the relevant marker gene in the pCRCT CRISPR plasmid. 3B. What would you expect to see if you plated on YED accidentally? Will most of the yeast be red or white? Why? 3C. The complete genotype of the host yeast strain is MATa ade2 his3 leu2 met15 ura3. Name one advantage of using a yeast strain with auxotrophies for several genes (i.e. his3, leu2 and ura3). 3D. After successful CRIPSR editing, the yeast can later be “cured” of the recombinant CRISPR plasmid – that is, the plasmid is lost but the CRISPR edit is stably inherited. Write the new genotype of the yeast-based on the ADE6 gene.
Competent E. coli cells were transformed with the pGLO plasmid. These transformed cells were then allowed to grow on two different plates: 1) a plate containing LB/AMP and 2) another plate containing LB/AMP/ARA. In which plate would you observe both phenotypic and genotypic changes? Briefly justify your answer. Edit View Insert Format Tools Table 12pt v Paragraph v BIUA e T?v 田 D2
7) Why are the following reagents used? Neutralizing solution (Plasmid isolation) Isopropanol (Plasmid isolation) RNase (isolation of genomic DNA)
Please answer the following using the experiment data below. Provide the formulas or methods used for calculations of each. Number of colonies on LB/amp/ara plate = Micrograms of DNA spread on the plates = Transformation efficiency = DNA plasmid concentration: 0.08 μg/μl 250 μl CaCl transformation solution 10 μl pGLO plasmid solution 250 μl LB broth 100 μl cells spread on agar 227 colonies of transformants
Drag and drop the appropriate text in the gaps below. Firstly, the plasmid DNA in the E. coli is propagated through Then the bacterial cells are pelleted by centrifugation at 10,000 RPM. The media supernatant is removed and the bacterial cells are The bacterial cells are then lysed via |. Contaminating macromolecules such as protein and chromosomal DNA is removed by Overnight incubation of the bacterial culture washed in cell resuspension buffer addition of lysis buffer addition of neutralisation buffer washing of the spin column separation via agarose gel electrophoresis
A target gene for producing human insulin must be inserted into a plasmid before the transformation process. Why the plasmid is important in this process? * I The target gene lacks the ability to replicate itself. II Screening process will be difficult without cloning vector. II The host cell is able to accept the recombinant plasmid but not the non- recombinant plasmid. O I and Il only I and III only O Il and III only O I, Il and III

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What is the correct order to prepare pure recombinant proteins when using E coli expression system? 1. Select appropriate column chromatography to purify the recombinant proteins