The pCR4-TOPO plasmid used in the lab has 3956 bp and contains two EcoRI sites flanking the cloning site. The PCR product of the ACTB gene used for the ligation was ~2000 bp. After transforming and selecting the cells, a liquid LB media was grown form a colony overnight and the plasmid isolated from the cells. Thereafter the plasmid was digested with EcoRI and ran in an agarose gel. What approximate band sizes did you expect to see in the gel? 1 band of ~6000 bp 4 bands each of ~1500 bp 3 bands of ~1000 bp, ~2000 bp, and ~4000 bp 2 bands, one of ~2000 bp and one of ~4000 bp I expect to see no bands on the gel

If an enzyme makes a single cut on a plasmid, how many bands will you see on the gel?fourthreetwoone

Describe and explain fully what you observe in Lane 5 of the agarose gel. Why is this samplenecessary?Determine the size of the linear bands in Lane 6 -8 using the graph paper provided. You mustshow all your calculations and hand in the graph generated.Do the bands in Lanes 6-8 of Figure 1B correspond with what you would expect for theserestriction digestions of pGEX4T-1? Explain your answer in full, with reference to the plasmidmap in Figure 1A and the agarose gel in Figure 1B and show all your calculations (you mustcompare your predicted band sizes to the calculated sizes from question 2i above).(3)(10)(8)40a)b)c)d)e)f)g)h)i)Question 3Panel C of Figure 1 on page 4 shows the results of an induction study and affinity purificationof the 26 kDa Schistosoma japonicum GST protein. The protein was induced by the addition ofIPTG to a final concentration of 1 mM and then purified using affinity chromatography. Answerthe following questions based on Figure 1, Panel C.If a stock solution of 2.5 M IPTG was used for induction, calculate the volume of stock solution(in μl) that would be added to a 50 ml culture to obtain the required final concentration. Youmust show all your calculations.What is the purpose of including the sample in the lane marked “UN”?What is represented by the samples labeled “P” and “CL” on the SDS-PAGE gel? Your answermust refer to the different fractions obtained after centrifugation and what is contained in eachof these fractions.Which molecule was bound to the resin used in the affinity purification of GST? Why is thisparticular molecule used to purify GST?Briefly describe the experimental procedure carried out to generate the sample shown in the lanedenoted “W” and explain why you would perform this step.What ligand was included in the elution buffer when you carried out this experiment, why wasit used and what kind of elution strategy does this represent?Explain how you would calculate the size of the eluted protein, with reference to one of the otherlanes on the gel. NOTE: you do not need to generate a graph or calculate the size, only explainHOW you would do this.Describe fully whether you think that the purification of recombinant GST was successful. Inyour answer, refer to the lane denoted “E” in Panel C.What additional experiment could you conduct to confirm that the purified protein is, in fact,GST?(3)(2)(4)(3)(3)(3)(3)(3)(1)25

A circular plasmid has three different but unique restriction sites for enzymes‘a’, ‘b’ and ‘c.’ When enzymes ‘a’ and ‘b’ are used together, two fragments ofequal size are generated. Enzyme ‘c’ creates fragments of equal size only fromone of the fragments generated by those cleaved by ‘a’ and ‘b’. The plasmid istreated with a mixture of ‘a’, ‘b’ and ‘c’ and analysed by agarose gelelectrophoresis. The number of bands observed in the gel is

f after running and analyzing the PCR products you found that all reactions including the positive control have no product or bands on the gel. What would be your conclusion? The human ACTB is unnecessary for the PCR The human ACTB sample used was too old The PCR was successful The PCR did not work The first step of the PCR should be avoided

What technique was used in the lab to analyze the products of the PCRs? Gel filtration chromatography SDS-PAGE Agarose gel electrophoresis Lipofection Transformation