Decide on two restriction sites that you can use to clone this into pL4440’s MCS. Identify their sequence. Tip: The plasmid map is in Figure 3, details of restriction site sequences can be found at https://enzymefinder.neb.com/#!#nebheader

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 + |

You have another circular plasmid. Complete and effective digestion of this plasmid with a restriction enzyme yields three bands: 4kb, 2kb, and 1 kb. In comparing the band intensity on an ethidium bromide-stained gel, you notice that the 4 kb and the 2 kb bands have the exact same brightness. The 1 kb band is exactly one fourth as bright as each of these. (Assume there is uniform staining with ethidium bromide throughout the gel.) How many times did the enzyme cut the plasmid? What is the size of the plasmid? Justify your answers to a and b above using a clearly labeled diagram showing the relative location of the cut-sites on the plasmid.

You utilised two plasmids in this practical, pOTC and pOTC-Δ. Plasmids are often represented using plasmid maps like the one below. This map shows the positions of recognition sites for a number of restriction enzymes Using the plasmid map of pBCH2.0 provided above, predict how many DNA fragments would be formed if this plasmid was digested with restriction enzymes EcoRI and PvuII.

Please look at the picture. Thanks

You are on a research project to study rice. Your want to use reverse transcription PCR to amplify the OsMEI28 cDNA from the plant and create a plasmid that contains the cDNA sequence in a vector. Briefly describe the steps that you would need to take.

In an restriction enzyme experiment where Eco RI and  Hind III are used . What would happen if a third restriction enzyme, BamHI were used in this lab if there was only one site on the plasmid that is recognized by BamHI? Explain what difference you would see in the gel

b. After molecular cloning and confirming you properly cloned stripes into the vector shown, you transform bacteria with the plasmid and select out bacteria that contain the plasmid. Now, you want to these transformed bacteria to express the STRIPES. Under what conditions would we need to grow bacteria transformed with this plasmid so that they express STRIPES? Explain your answer, and be sure to describe the important regulatory regions on the plasmid above and what interacts with these regulatory regions in the conditions you have indicated.

Draw a diagram showing what pGEM will look like after it has been digested with BamHI. Be sure to show both strands (with enzyme sequence) and include the origin of replication, the ampicillin resistance gene, 3’ and 5’, and the DNA sequence at the restriction site. It will probably be easiest to simply draw it as an open circle, not a line. Now show what pGEM will look like after it has been digested with EcoRI instead of BamHI. Again, be sure to show both strands and include the origin of replication, the ampicillin resistance gene, 3’ and 5’, and the DNA sequence at the restriction site.

A PCR reaction was performed to amplify the XULA4 gene, which is bp 524-6,480 on a plasmid that is 9,435 bp.  After the PCR, the product was digested with XhoI.  There are XhoI sites on the plasmid at bp 151, 1,336, and 4,795.  Calculate the size(s) that would result when the product is digested with XhoI.  Then enter the size of the largest fragment (in bp).