1) You wish to make a restriction map of a 17.0 kb linear fragment. You digest the fragment with Sbf1, Pst1, and a mixture of Sbf1 and Pst1.
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- A 2.0kb bacterial plasmid ‘BS1030’ is digested with the restriction endonuclease Sau3A; the plasmid map is depicted in the diagram below and the Sau3A (S) restriction sites are indicated. Which of the following DNA fragments do you expect to see on an agarose gel when you run Sau3A-digested plasmid ‘BS1030’ DNA? a. 250 bp, 450 bp, 550 bp, 1.1 kb, 1.5 kb and 2.0 kb b. 2.0kb c. 250 bp, 400 bp, 450 bp, 500 bp and 550 bp d. 100 bp, 200 bp, 250 bp, 400 bp, 500 bp and 550 bpA 12 kb linear DNA fragment is subject to single or double RE digest and agarose gelelectrophoresis, to yield the gel profile shown below. The first lane contains the size marker(M).a) Explain how the name of the enzyme EcoRI is derived.b) How many sites are there for EcoRI and PvuII respectively on this DNA fragment?c) Use the sizes of the DNA bands on the gel to compile a restriction enzyme map of the DNAfragment. Indicate the positions of the restriction enzymes sites for EcoRI and PvuII on themap.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 pairs
- You digest 4 uL of plasmid DNA that is 50 ng/uL concetration in a total volume of 20 uL. You run 10 uL of the digest on teh gel. You then do a DNA purification protocol with a Zippy prep on the remaining digested DNA. You elute the DNA in a 25 uL. A 2 uL ssample has the concentration of 2 ng/uL. What is the DNA yield?You will be setting up your diagnostic digest on three plasmids, the ones you began with, pGFPuv and PHSG298 and your presumptive pHSG298-GFP. Complete the table below: Solution Stock Working Volume for one reaction Volume for Master Mix (3.5X) Cutsmart buffer 10X 1X Restriction enzyme(s) 1 µl uL Water UL UL 2 µl 25 pL DNA Total volumeE 1 kb 1 kb DAK 6 kb Gene X E1 kb B 5 kb. Gene Z E₁kb 1 kb The figure above shows a linear chronosome containing Gene X and Gene Z and the location of sites for the restriction enzymes EcoRI (E) and BamHI (B). Refer to this map as you answer the question below. If you digest this DNA with EcoRI, then perform agarose gel electrophoresis, how many bands, and what sizes, would you expect to see? a) Two bands on the gel corresponding to the sizes 2 kb and 6 kb b) Three bands on the gel corresponding to the sizes 1 kb, 6 kb and 8 kb. c) Three bands on the gel corresponding to the sizes 1 kb, 5 kb and 6 kb d) Four bands on the gel corresponding to the sizes 1 kb, 2 kb, 5 kb and 6 kb e) Four bands on the gel corresponding to the sizes 2 kb, 3 kb, 4 kb and 8 kb
- Given the DNA sequence of the restriction enzyme: gi|6329444|dbj|AB034757.1| Hynobius retardatus mRNA for larval beta-globin, complete cds GCAGAATCTGACTCAAGAAATCCCTCCTCACCCAACACCACCAGCAGCCATGGTTCACTGGACAGCAGAGGAGAAGGCAGCCATCAGCTCTGTGTGGAAGCAGGTGAACGTGGAGAGCGATGGACAGGAGGCCCTGGCCAGGTTGCTGATCGTCTACCCCTGGACCCAGAGATACTTCAGCTCTTTTGGGGACCTGTCGAGCCCAGCTGCCATTTGTGCCAACGCCAAGGTCCGTGCCCATGGCAAGAAGGTCCTGTCCGCCCTGGGAGCCGGCGCCAACCACCTGGATGACATCAAAGGCAACTTTGCTGATCTGAGCAAGCTTCACGCAGACACACTCCATGTGGACCCCAATAACTTCCTGCTCCTGGCAAACTGCCTGGTGATCGTCTTGGCCCGCAAGCTGGGAGCCGCCTTCAACCCTCAAGTCCATGCGGCCTGGGAGAAGTTCCTGGCCGTCTCCACCGCGGCTCTGTCCAGAAACTACCACTAGAGACTGGTCTTTGGGTTTAATTCTGTGAACGTCCCTGAGACAAATGATCTTTCAATGTGTAAACCTGTCATTACATCAATAAAGAGACATCTAACAAAAAAAAAAAAAAAAAAAAAAAAAA Identify two blunt-end cutters Identify two sticky-end cutters. For each, Provide the sequence of the Restriction enzyme, Highlight using a specific color where the DNA sequence where the restriction enzyme will cut the DNA Indicate the…A piece of DNA 5.0 kb long is cloned and then cut out of the vector for analysis. This linear piece of DNA is digested with two restriction enzymes, EcoRI and BamHI, individually and in combination, and the resulting fragment sizes are determined by electrophoresis. The results are as follows: Restriction fragment size 4.5 kb; 0.5 kb 3.0 kb; 2.0 kb 2.5 kb; 2.0 kb; 0.5 kb Enzyme name EcoRI ВатHI EcoRI + BamHI Construct a potential restriction map based on these results.If a 500 bp of DNA between the two restriction sites were deleted, how would the banding pattern on the gel differ from the one you drew in part a? (Part a is attached)
- A small DNA molecule was cleaved with several different restriction nucle- ases, and the size of each fragment was determined by gel electrophoresis. The following data were obtained. Enzyme Fragment Size (kb) EcoRI 1.3, 1.3 Hpall 2.6 HindlII 2.6 ЕcoRI + Hpall ECORI + HindlII 1.3, 0.8, 0.5 0.6, 0.7, 1.3 (a) Is the original molecule linear or circular? (b) Draw a map of restriction sites (showing distances between sites) that is consistent with the data given. (c) How many additional maps are compatible with the data? (d) What would have to be done to locate the cleavage sites unambiguously with respect to each other?A circular plasmid of 10,000 base pairs (bp) is digested with two restriction enzymes, A and B, to produce a 3000 bp and a 2000 bp bands when visualized on an agarose gel. When digested with one enzyme at a time, only one band is visible at 5000 bp. If the first site for enzyme A (A1) is present at the 100h base, the order in which the remaining sites (A2, B1 and B2) are present is - (A) 3100, 5100, 8100 115. (B) 8100, 3100, 5100 (C) 5100, 3100, 8100 (D) 8100, 5100,. 3100A group of overlapping clones, designated A through F, is isolated from one region of a chromosome. Each of the clones is separately cleaved by a restriction enzyme, and the pieces are resolved by agarose gel lectrophoresis,with the results shown below. There are nine different restriction fragments in this chromosomal region, with a subset appearing in each clone. Using this information, deduce the order of the restriction fragments in the chromosome.