Lab 7 & 8 Assignment
BIMM 101: Recombinant DNA Lab
Lab 7 & 8 Assignment
Part B
1. A genomic library is a “collection of recombinant vectors or clones, among which is representative of the entire genome of the organism” (BIMM 101 Lab Manual, 47). In order to create a genomic library, genomic DNA from Vibrio fischeri was first isolated then treated with Sal I restriction enzyme to generate inserts (smaller fragments of DNA). Sal I restriction enzyme was also used to treat the vector plasmid in order to digest the V. fischeri DNA fragments. The inserts and the vector were then ligated together. E.Coli cells were then made competent in order to take up the plasmid DNA by transforming these competent cells with a “ligation mixture to create a population of host bacteria containing different combinations of the ligated inserts and vector” (BIMM 101 Lab Manual, 46). A colony indicated that the cell had taken up the vector. Whether or not that colony contained the genes of interest was determined by screenings such as antibiotic resistance, blue-white color screening, and luminescence. A bioluminescent colony immediately indicated the desired genes and where re-streaked while the plasmids of non-glowing white colonies where further isolated and sequenced for the desired genes.
2. A. The L1-L4 ligation reactions all contained varying amounts of the digested V. fischeri (insert) DNA, 2 microliters of digested pGEM, varying amounts of water, and 6 microliters of 5X ligation buffer. Samples were made with varying amounts of insert to vector ratio to test which ratio would yield the greatest results. The idea behind this is that if there is a higher number of insert to vector, there are greater chances of an insert binding. Thus, a higher ratio of insert to vector is ideal. A chart is provided below.
Tube Insert to Vector Ration Digested V. fischeri (insert) DNA Digested pGEM H2O 5X Ligation Buffer Total Volume
L1 2:1 0.30ug = 6.25µl 0.05ug = 2µl 15.75µl 6µl 30µl
L2 3:1 0.45ug
Lab 7 & 8 Assignment
Part B
1. A genomic library is a “collection of recombinant vectors or clones, among which is representative of the entire genome of the organism” (BIMM 101 Lab Manual, 47). In order to create a genomic library, genomic DNA from Vibrio fischeri was first isolated then treated with Sal I restriction enzyme to generate inserts (smaller fragments of DNA). Sal I restriction enzyme was also used to treat the vector plasmid in order to digest the V. fischeri DNA fragments. The inserts and the vector were then ligated together. E.Coli cells were then made competent in order to take up the plasmid DNA by transforming these competent cells with a “ligation mixture to create a population of host bacteria containing different combinations of the ligated inserts and vector” (BIMM 101 Lab Manual, 46). A colony indicated that the cell had taken up the vector. Whether or not that colony contained the genes of interest was determined by screenings such as antibiotic resistance, blue-white color screening, and luminescence. A bioluminescent colony immediately indicated the desired genes and where re-streaked while the plasmids of non-glowing white colonies where further isolated and sequenced for the desired genes.
2. A. The L1-L4 ligation reactions all contained varying amounts of the digested V. fischeri (insert) DNA, 2 microliters of digested pGEM, varying amounts of water, and 6 microliters of 5X ligation buffer. Samples were made with varying amounts of insert to vector ratio to test which ratio would yield the greatest results. The idea behind this is that if there is a higher number of insert to vector, there are greater chances of an insert binding. Thus, a higher ratio of insert to vector is ideal. A chart is provided below.
Tube Insert to Vector Ration Digested V. fischeri (insert) DNA Digested pGEM H2O 5X Ligation Buffer Total Volume
L1 2:1 0.30ug = 6.25µl 0.05ug = 2µl 15.75µl 6µl 30µl
L2 3:1 0.45ug