The following plasmid is digested with EcoR1 and Ndel restriction endonucleases. Agarose electrophoresis is then used for analysis. How many bands do you expect to see on the agarose gel after complete digestion? oic 6000-- EcoR1 8000 bp FR1 Bank Answer: Explanation: Ndel 4000
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- 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 + BamHIA result of agarose gel electrophoresis of plasmid DNA is shown below. The direction of the electrophoresis is indicated by the arrow head. Lane 1 shows the plasmid DNA with no restriction enzyme digestion. Lane 2 shows the plasmid DNA which has been digested by restriction enzyme ECORI. Given the fact that there are only relaxed and supercoiled forms existing in this plasmid, please annotate the bands in Lane 1 on this gel with their supercoiling status (supercoiled or relaxed) and explain why. Then based on the information from Lane 1 and Lane 2, analyze the number of EcoRI cleavage sites on this plasmid. Lane 1 2Competent 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
- U have the plasmid pUC18/19, which is a circular plasmid that consists of 2686 bp. What would the number of and length of the fragments be if you cut the plasmid with the following restriction enzymes or combination of enzymes? Give a schematic representation of the digestions. PscI & GsuI ______________________________________________________________ ScaI, PdmI & BsaXI ______________________________________________________________ ScaI, SspI & EheI ______________________________________________________________You wish to insert a gene in the plasmid below. (Note the neon green lines represent restriction sites.) amp gene BamHI Sacl Saçl EcoRI BamHI 33 BamHI Gene of interest Chromosomal DNA from human cells a) Which restriction enzyme should you use to cleave the plasmid? Explain your answer. b) How would you create a cDNA library for the chromosomal DNA containing the gene of interest? c) The green area on the plasmid is the ampicillin resistance gene. Explain how you would use it to screen for a recombinant plasmid. 1Which of the following scenarios would ONLY occur if your skipped the digest purification step? UV/VIS spectrophotometric quantification of DNA may be skewed by uncut plasmid. Some plasmid molecules may be cut once, by one enzyme, and re-ligate to themselves. The fragments cleaved by the restriction enzymes on the plasmid and insert can re-ligate to their sites, causing reduced ligation efficiency. Plasmid molecules cut with EcoRI and Xbal can ligate to each other instead of the insert.
- A cloning vector map is shown below. EcoRI Bam Ban Hind P-galactosidase Amp Bam Bam EcoRI Ori C Which restriction site is best for inserting a DNA fragment for selection of chimeric plasmid containing colonies? 1) They're all equally good. 2) Hindll 3) EcoRI 4) BamHIDraw the gel electrophoresis results for the following 16 kb plasmid when the plasmid is cut withrestriction enzyme A, with restriction enzyme B and with both A and B restriction enzymes. The Aenzyme cuts at positions 0 and 9.2 kb and the sites are shown as white. (0 is at the top of the circle.) TheB enzyme cuts at positions 3.8, 7 and 13.2 kb and the sites are shown as gray. Show the electricalpolarity of the gel, the bands, and their sizes in your drawing. Make sizes consistent between gel lanes.Explain why the concept of charge density is important for gel electrophoresis.A plasmid is cleaved by restriction endonucleases and analyzed by agarose gel electrophoresis. Assume there is no supercoiling. Answer the following three questions about the plasmid. a. What is the size of plasmid? Explain briefly. b. How many HindIII sites are there in the plasmid? c. Is it possible to determine that how many EcoRI cut sites available in plasmid? Why? 1 2 3 4 Lane 1: DNA ladder (100 bp increments beginning at 100 bp), Lane 2: EcoRV, Lane 3: Smal, Lane 4: HindIII
- In the "Bacterial Transformation" experiment, why were there no fluorescence bacteria present in the other plates even though these were inserted by the +pGLO plasmid? (Note: Discuss the importance on the addition of ampicillin and arabinose to the medium of the genetically engineered bacteria). Limit your answer to 1-3 sentences only.Below is a plasmid with restriction sites for Baml and EcoRI, Several restriction digests were done using these two enzymes either alone or in combination. Hint: Begin by determining the number and size of the fragments produced with each enzyme. "kb" stands for kilobases, or thousands of base pairs. Plasmid Gel lanes I | III IV V 6 Kb Bam HI Bam HI. 20 Kb PGEN101 (20 Kb) 2 Kb 11 Kb Bam HI 8 Kb 8 Kb 4 Kb 6 Kb Eco RI 3 Kb 21. Which lane shows a digest with BamHl only? a. I b.I c. II d. IV e. V 22. Which lane shows a digest with EcoRLonly? a. I 23. Which lane shows the fragments produced when the plasmid was incubated with both EcoRI and BamH1? a. I b.I c. II d. IV e. V b. I c. II d. IV e. V Base pairsThe map of plasmid pUC19 is shown below. The restriction site coordinate is the position of the 5’base on the top strand of each site sequence. The restriction enzyme sites are in bold type if there is only one site in pUC19. Please list the fragments in order of size, largest to smallest, which will result from a complete digestion by the restriction enzyme PvuII. Please list the fragments in order of size, largest to smallest, which will result from a complete digestion by the restriction enzyme DrdI.