very lost on this whole worksheet and i dont know if my current answers are even...
Can someone please help me answer these? Neat work is greatly appreciated. Explanation will also be appreciated (optional, if you have time). Thank you so much! I will give a big thumbs up :) (1) You start with the pUC18 plasmid that is a -2.7 kb circular DNA molecule. The multiple cloning site (i.e., polylynker) has unique restriction sites in the following order: HindIII, BamHI, EcoRI. Recall that unique restriction site means that these sites are only found once on...
Question 2,3 and 4 aBbCcDdE AaBbCoDdE Normal No Spacing BamHI 300 EcoRI 3400 1000 2000 5 kb EcoRU Apal List three components a cloning vector must contain A cloning site A replication origin A selectable marker -Drug resistant gene 1. 2. Draw the end of the fragment that will be generated after digestion with the Apal enzyme. Indicate the type of ends generated by this enzyme (Blunt/Sticky, 5' or 3' overhang) (3) 3. What is the size of the plasmid...
III. Subclone the gene into plasmid, extract the plasmid DNA. 5. You know that your insert (gene of interest, GOI) is flanked by the EcoRI sites, which makes this restriction enzyme a perfect candidate to cut out your gene. You also know that the GOI has a unique BamH1 restriction site. After subcloning the PCR product into the plasmid, a purified DNA preparation of the plasmid is digested to completion with BamHI restriction endonuclease. In separate reactions, the same preparation...
Question 4 C. Cloning and restriction enzyme digest Video aid 1: Plasmid cloning Video aid 2: Restriction enzyme digest analysis The PCR was a success and your target region of 440 bp in length has been amplified. You igate a short linker containing an Apal restriction enzyme site onto both ends of the PCR product, digest it with Apal, and clone it into the Apal site of the 5 kb plasmid diagrammed below Bamll 300 EcoRI 3400 1000 2000 5...
3. With reference to your PCR amplicon, detail the terminal DNA sequences at the two ends of the fragment that will be generated after digestion with Eael (indicate these in the two blocks below). What type of ends are generated by this enzyme (blunt/sticky, 5' or 3 overhang)? (4) GGCCR PCR amplicon RCCGG The PCR was a success and your target region of 770 bp in length has been amplified. You now plan to digest the DNA amplicon with the...
1: EcoRI 2: BamHI 3:PstI 4:EcoRI + BamHI 5:EcoRI + PstI 6:BamHI + PstI 7: EcoRI + BamHI + PstI Draw a restriction map of this plasmid. To determine the restriction map of a plasmid, you digest it with one or more of the three restriction enzymes EcoRI BamHI and PstI. Sfter digestion, you anslyzr the size of the fragments obtained on electrophoresis gel and you obtain the following gel piste1: EcoRI: 5400 bp piste2: BamHI: 5400bp piste3:PstlI: 4400bp ,...
Cloning / Subcloning When subcloning engineering new plasmids, by inserting new DNA fragments (inserts) me plasmids (now called a vector because it will carry your gene of interest) it is important to considering existing genes / DNA elements. If a site is in the middle of a gene, you could lose or destroy that gene. If there are multiple sites for an enzyme, when you paste them together, multiple possible outcomes can arise. This is undesirable, because it confounds verification...
7. Explain the procedure for cloning DNA fragment into the plasmid PBR322 (shown on the right) (S pts.). The gene fragment of interest was obtained by digestion of chromosomal DNA with the restriction enzyme Sall and subsequent purification using agarose gel electrophoresis. Which antiblotic would you use in the final step of the cloning procedure, and Pst why? EcoR Sal Ampicillin Tetracycline resistanica(Ter Amp) PBR322 4,361 bp) Origin of replicatiorn (ori Pvull 8. Assume that your gene fragment from question...
You are performing an analysis of squirrel mitochondrial DNA, which is a circular double-stranded DNA molecule that is 23,000 bp in length. You are using restriction enzymes and agarose gel electrophoresis in your experiments. You have decided to use two restriction endonucleases: Pstl and Tagl. The picture below shows their recognition sites, and the red arrows indicate their strand- Pstl (Providencia stuartii) CTGCAG GACGTC specific cleavage sites. Taqi (Thermus aquaticus) TCGA AGCT Part A: The Pstl and Taql enzymes both...
A1. The following is the DNA sequence of a hypothetical gene for the SMALL protein. It is called the SMALL gene. i atgggattac actgtcacga ccaaatagcc ttcattgtat 41 caaaaggato aatcgagtta tag Imagine you are doing a research project in a laboratory and your supervisor asks you to clone the SMALL gene into the PBR322 plasmid (shown below). You must use the Pstl and EcoRI sites for your cloning. HindIII EcoRI | EcoRV BamHI 4359 0 29 185 4000 375 Sall Psti...