Lab 6: Isolation Of Chromosomal DNA
Lab 6: Isolation of Chromosomal DNA
Mic 428L/ Section 001
Introduction: In biological research to address and eventually answer a multitude of questions, usually involves isolating chromosomal DNA. The purpose in this particular lab was to isolate chromosomal DNA from mutants grown and observed in lab 5 and then digest the DNA using a restriction enzyme. The fragments left from digestion will be ligated and then transformed into a strain of E. Coli DH5αλpir containing the pir gene pi product for replication. This gene will allow only the plasmid containing the Tn to replicate. Afterward, the plasmid was isolated and sequenced into adjacent chromosomal DNA to determine which gene was interrupted by the Tn.
The isolation of genomic …show more content…
22μl will then be added to a chilled electroporation cuvette. The cells will be electroporated using a Bio-Rad Gene Pulser, which will send electrical pulses through the E.coli so that the exogenous DNA can enter it. Room temperature SOC buffer is then added to stabilize the cells after being in a state of stress from the electroporation. The solution is then transferred to a new 1.5 ml microfuge tube and stored at 37°C for 60 minutes. 200μl of the solution will then be spread on an LB+Kan50 plate to select for the plasmid that contains the kanamycin resistance marker. These plates will be incubated at 37°C for 1-3 days. After 1-3 days, we will select 2 colonies from each plate and retouch a new LB+Kan50plate in order to ensure that we obtained the plasmid DNA.
Isolation of plasmid DNA for sizing and sequencing
The isolated plasmid DNA will then be run on an agarose gel to determine the size of it. Tracking dye will be added to the solution which contains glycerol that will weigh the samples down in the wells.
Mic 428L/ Section 001
Introduction: In biological research to address and eventually answer a multitude of questions, usually involves isolating chromosomal DNA. The purpose in this particular lab was to isolate chromosomal DNA from mutants grown and observed in lab 5 and then digest the DNA using a restriction enzyme. The fragments left from digestion will be ligated and then transformed into a strain of E. Coli DH5αλpir containing the pir gene pi product for replication. This gene will allow only the plasmid containing the Tn to replicate. Afterward, the plasmid was isolated and sequenced into adjacent chromosomal DNA to determine which gene was interrupted by the Tn.
The isolation of genomic …show more content…
22μl will then be added to a chilled electroporation cuvette. The cells will be electroporated using a Bio-Rad Gene Pulser, which will send electrical pulses through the E.coli so that the exogenous DNA can enter it. Room temperature SOC buffer is then added to stabilize the cells after being in a state of stress from the electroporation. The solution is then transferred to a new 1.5 ml microfuge tube and stored at 37°C for 60 minutes. 200μl of the solution will then be spread on an LB+Kan50 plate to select for the plasmid that contains the kanamycin resistance marker. These plates will be incubated at 37°C for 1-3 days. After 1-3 days, we will select 2 colonies from each plate and retouch a new LB+Kan50plate in order to ensure that we obtained the plasmid DNA.
Isolation of plasmid DNA for sizing and sequencing
The isolated plasmid DNA will then be run on an agarose gel to determine the size of it. Tracking dye will be added to the solution which contains glycerol that will weigh the samples down in the wells.