You are studying a new plasmid, and you digest the plasmid with three restriction enzymes: Eco RI (E), Hindll (H), and Xbal (X). You digest the plasmid DNA with each of the following combinations of enzymes and observe the results on an agarose gel. You are provided a partial plasmid map as shown below to the right.

EDTA weakens the cell wall by removing ions that help hold it together while glucose prevents premature cell lysis due to osmosis in cells with weakened cell walls. You have made a batch of resuspension solution to isolate plasmid DNA, but you forgot to add EDTA. What do you think will happen when you try to perform a plasmid isolation procedure with this reagent? Why?

The plasmid cloning vector pBR322 is cleaved with the restriction endonuclease PstI. An isolated DNA fragment from a eukaryotic genome (also produced by PstI cleavage) is added to the prepared vector and ligated. The mixture of ligated DNAs is then used to transform bacteria, and plasmid-containing bacteria are selected by growth in the presence of tetracycline. The cloned DNA fragment is 1,000 bp long and has an EcoRI site 250 bp from one end. Three different recombinant plasmids are cleaved with EcoRI and analyzed by gel electrophoresis, giving the patterns shown below.   What does each pattern say about the cloned DNA?   Note: pBR322, the PstI and EcoRI restriction sites are about 750 bp apart. The entire plasmid with no cloned insert is 4,361 bp. Size markers in lane 4 have the number of nucleotides noted.

Shown below is a diagram for a plasmid vector you want to use to clone a gene. The diagram shows the location of the recognition sites for four restrictions enzymes, BamHI (B), EdoRI (E), Hindill (H), and Xhol (X). The genes encoding beta-lactamase (AmpR) and beta-galactosidase (lacZ) are indicated. If you were to use this vector, which enzyme should be used to linearize the plasmid in preparation for cloning? E B lacz O Hindi!! BamHI O EcoRI O Xhol H EcoRI and Xhol E -X AmpR

Which 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.

Margaret has been given a plasmid containing her favorite gene, afg (Margaret’s favorite gene). The only thing sheknows is that afg was cloned into the vector using a single restriction enzyme, BstBI. Margaret orders theBstBI enzyme from NEB, so she can cut the plasmid and confirm the presence of the insert. On theNanodrop, she measures the concentration of her plasmid to be 401 ng/μL. Describe how she would setup and perform this reaction if she wanted to digest 2.0 μg of the plasmid.

A DNA sequence is shown below, which includes a gene as marked. You have the restriction enzymes SalI and HindIII available to you to excise the gene prior to its incorporation into a plasmid vector. Which would you use to excise the gene?

In an attempt to clone a gene into the TOL plasmid, it was observed that the restriction product of the gene of interest were blunt ended, thereby reducing the ligation efficiency of this gene and the TOL plasmid. Elucidate two methods that could be employed to increase the ligation efficiency of your restriction products.

true or false if a restriction enzyme recognizes the restriction site, 5' AACGTT3', and the enzyme cuts between the second A and the C, this will produce a "sticky end," which is useful for cloning into a plasmid vector.

PCHEM4321. An agarose gel electrophoresis pattern of the plasmid PSPM4321 digestion (restriction) is shown below. Draw a restriction map of a plasmid with the appropriate restriction sites based on the data given below. Hindlll Hindll BamHI +BamHI Figure 1: 1% agarose gel electrophoresis of pCHEM4321 40 24 16 12 12 8 4 4 + |