Biology 475 Molecular Biology Lab Two – Library Screening with Restriction Enzymes Elizabeth Jacobsmuhlen Spring 2003

Introduction To use the mixed population of genes (Red & Green16S) that were amplified using PCR last week to isolate plasmids via cloning with E. coli (cloning was done for us by Dr. Boomer and Danny between Lab One and Lab Two). The cloned E. coli colonies will be used to isolate plasmids via the use of rapid boiling mini-prep plasmid isolation methods. Those plasmids will then be screened via the use of an electrophoresis gel. Finally, a “macro-array” of these plasmids will be started that will be finished week #3.

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	Cloning Results
Results: This is the library of clones grown on ampicillin and X-Gal media. On this plate is growing all of the E. coli that have taken up plasmids. The plasmids may or may not have taken up an insert. The E. coli need to be screened using a feature of X-Gal media that allows the distinction between plasmids without insert (blue) and plasmids with insert (white).

	Mini-Prep Methods
Methods: Before this step the samples were centrifuged and DNA and cell fragments removed by dumping off the supernataunt. The samples were then resuspended using buffer, lysozyme added, vortexed, boiled, and then centrifuged. The picture to the right shows the resuspension with buffer step.
Methods: After the samples were taken out of the centrifuge the snot pellet was removed (shown to the right). This pellet should contain everything but plasmids. The plasmids will be used for digestion/restriction and electrophoresis procedures later in the lab.

	Restriction Methods and Results
Digestion Methods: Samples were put into a hot water bath (37 degrees Celsius for 45-90 minutes). This incubation step allows the Eco RI to cut the plasmid and allows the insert to be removed. This temperature is used because it is the optimum temperature Eco RI to digest or cut out the insert from the vector.
Electrophoresis Methods: This picture to the right is of the electrophoresis apparatus. DNA is loaded into an agarose gel and the equipment is hooked up to an electrical current. This set up pulls the negatively charged DNA to the positive end through the use of the electrical charge. As the DNA moves it is separated by molecular size/weight due to the composition of the agarose gel.
Results: Lane 1: Molecular weight standard Lane 2: Clone #71 Lane 3: Clone #72 Lane 4: Clone #73 Lane 5: Clone #74 Lane 6: Clone #75 Lane 7: Clone #76 Lane 8: Clone #77 Lane 9: Clone #78 Lane 10: Clone #79 Lane 11: Clone #80

Discussion

This picture shown above in the results section is of the electrophoresis gel after being treated with ethidium bromide and placed under UV light. Lane 2 and 3 show plasmid only with no insert. Lane 4, 6, 9, and 11, most likely are red bacteria DNA insert and plasmid. Lane 5 and 10, most likely, show green bacteria DNA insert and plasmid, because there is the primer band (very top) as well as two insert bands.

This procedure worked well. The DNA snot pellet formed and was easy to remove which indicated that it was boiled and centrifuged for the proper amount of time. The electrophoresis gels showed that both primer and insert DNA were present. Also in the case of the green insert which has an Eco RI site in it the insert it is cut into two pieces by the Eco RI. This is shown in the electrophoresis gel shown above. In the case of my gel both green and red DNA are most likely present. The red DNA insert is merely cut out of the plasmid at its ends by Eco RI and therefore shows as a primer and one DNA fragment band on the gel. This experiment is used to show that the insert and primer were present and these samples can be used in later labs for further analysis/identification. This lab allowed us to gain a better idea of which clone samples contained red or green bacteria.