LAB REPORT: EXPERIMENT 4 BIOC 433 LAB L. RESTRICTION DIGESTION AND TRANSFORMATIO
ID: 145311 • Letter: L
Question
LAB REPORT: EXPERIMENT 4 BIOC 433 LAB L. RESTRICTION DIGESTION AND TRANSFORMATION II. MEASURING PROTEIN CONCENTRATION Part I: 1. Answer the following questions: a) Why does this cloning experiment yield both blue and white colonies? b) Why are there two different sticky ends on the PCR amplified DNA? c) What is a diagnostic step to analyze the success of the various steps in this experiment? 2. Using the plasmid vector Ssp t 2142 Aat 1 1937 shown here, outline your strategy and steps for cloning gene X. 2260 Scal 1818 Noe l T7 star NarI 2561 Sacl Sma23 BamHI 20 Xa 32 pGEM -3Z (2743bp) Acc 39 Hinc40 Sohl 54 ind 56 69 ori Part 1. Present the data for each determination by UV or Bradford in graphical and tabular form. 2. Determine protein concentration of the unknowns by each method. 3. How do these values compare with one another? Supplemental Information Asking to determine protein concentration of the unknowns by UV Absorption at 280 nm. The table contain calculated BSA] We should be able to read unknown concentration from the graph. A graph should have concentration on x-axis and absorption on y-axis. Tube # | BSA (1000 g/mL) Water Volume [BSA], (ug mL)Aso Volumn 0 AL 250 500 uL 750 1000 uL Unknown #1 (1 mL) Unknown #2 (1 mL) 1000 750 uL 500 L 250 0 Hg/mL 250 ug/mL 500 Hg/mL 750 g/mL 1000 g/ml. BLANK 0.141 0.288 0.445 0.587 0.232 0.399 Supplemental Information Asking to determine protein concentration of the unknowns by Bradford Assay The table contain calculated {BSAL We should be able to read unknown concentration from the graph. The graph should have concentration on x-axis and absorption on y-axis. Tube # | BSA (1000 g/mL) | Water | Bradford | BSA (ug/mL) | Ase Volume Yolnme Reagent (5x) 800 L 200 HL 795 200 790 200 780 HL 200 HL 770 | 200 | Unknown #1 (20 ) | 780 200 | Unknown #2 (20pl) | 780 ! 200 0 /mL BLANK 5.03 /mL | 0.333 10.1 ug/mL 0.564 20.41 g/mL | 1.146 30.93 ug/mL 1.424 0.632 0.875 310 L | 20pL 5 30 HL 6 7Explanation / Answer
Part I
1a. Not all the plasmid sucessfully insert and ligate the gene of interest during the ligation step in a molecular cloning experiment. As a result, transformation of bacteria with this mixture of plasmids may or may not produce the intact - galactosidase gene to convert the substrate, X-gal into blue colored product yielding both blue and white colonies.
1b. Two sticky ends ensures that the gene of interest is inserted in into the plasmid in a correct orientation during the ligation step in a molecular cloning experiment which in turn ensures the expression of the recombinant gene.
1c. A Polymerase Chain Reaction (PCR) with an approproate pair of primers followed by restriction digestion with an appropriate pair of enzmes ensures the success of each step in a cloning experiment.
2. Step1: Amplification
Amplify gene X using PCR with the primers designed to ensure the inclusion of two different restriction sites corresponding to two different restriciton enzyme in the MCS region of the plasmid.
Step 2: Restriction digestion
Linearization the plasmid and production of sticky ends in the amplifies gene by digesting the DNA with the combination of restriction enzymes.
Step 3: Ligation
Ligation step ensures the insertion of the gene X into the linearized plasmid.
Step 4: Transformation of bacteria with recombinant plasmid.
Step 5: Screening for Blue- white colonies.
Part II.
1. Data is already present in tabular form. Let me help you to analyse the data.
2. If we notice the data from UV absorption experiment:
Unknown#1 has a UV absorption of 0.232 which is typically between the values obtained for 250 ug/ml (0.141) and 500 ug//ml (0.288) standard. So when we plot the values in a graph paper and check the corresponding values, the concentration of the unknown prortein will come somewhere between these two concentrations.
Similarly for unknown#2 the concentration of pretein will be between 500ug/ml and 750ug/ml as the OD 0.399 lies between 0.288 and 0.445
Similarly for Bradford assay:
Unknown1: Protein concertration is between 10.0 ug/ml and 20.41 ug/ml.
Unknown2: Protein concertration is between 10.0 ug/ml and 20.41 ug/ml. Note here as the OD for unknown 2 is higher than unknown 1, the protein concentration will be more for unknown 2.
3. Bradford assay is a very sensitive assay when compared to UV absorption method for proetin estimation. Bradford assay can detect and quantitate the protein concentration as low as 1-20 ug which the UV absorption method fail to detect.
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