Biology 475

Molecular Biology

Lab Two – Library Screening With

Restriction Enzymes

Andy Mikles

2003

Introduction In our first lab, we isolated 16s genes from microbial mat bacteria. Since then, they had been put into vectors and grown in E. coli. In this lab, our goal was to isolate those vectors from E.coli which had taken up inserts, we would then take those samples, and run gels on them, and also make western blots in order to study them in more depth in lab 3. (that’s me preparing the blot)

Cloning Results

Results: This is a picture of our clone library. Each dot represents and individual colony of E. coli. These colonies grew on a media of ampicillin and lac. We took these individual colonies and grew them in media of test tubes which contained ampicillin. The resulting E. Coli were almost certain to contain our inserts. These test tube colonies are what we used for our minipreps for electrophoresis and blots.

	Mini-Prep Methods
Methods: isolating the vectors from E.coli, we started with the amp media in test tubes. I had samples from colonies 1-10. we pelleted the cells from these tubes, and used similar techniques from lab one to isolate the plasmids.
Methods: After we pelleted the plasmids in our tube, we dried them and resuspended them in TE. Which kind of cleaned our sample for better results when we do electophoresis.

	Restriction Methods and Results
Digestion Methods: Once we had our vectors isolated, we could use restriction enzymes to cut them into pieces. We used EcoR1 which would cut on either side of our insert, and depending on the bacteria, it might cut somewhere in our insert as well. Here, norm is adding our cocktail to his vectors.
Electrophoresis Methods: Cutting the vectors into pieces allows us to separate them on gels in order to identify differences in our samples. Here, we loaded a 1% agrose gel with samples and ran them for 45 min. @125 volts.
Results: Here is a picture of the electophoresis. Lane 1 one the bottom is the standard, and all the lanes that have 2 bands have an insert. The first band in each row is a vector that got cut by ecor1. the second line if present is the length of the vector.

Discussion

In this lab we took the cloned E.coli that had our PCR insert in them, and then tried to isolate the vectors in order to a) make blots for hybridizing (see lab 3) and b) run gels on them to see if they took up inserts. In order to isolate our vectors, we first pelleted our cells to concentrate them out of the media, then we resuspended them in STET which disrupts the cell wall and also added lysozyme which digests the cell wall, this will lyse the cell, so we then boiled them to bind proteins and clump the samples. At this point, vectors which are small and unbound should be floating in the solution. So we centrifuged this in order pellet the cell parts so they could be removed. The pellet cam out nicely and was easily removed. We then added salt and alcohol to precipitate the plasmids and then centrifuged them to pellet. I did not really see a pellet but was assured that there was product there and to go with it. We then cut the vectors with ecor1 and incubated them to cut them into chunks, and loaded them into a 1% agrose gel, which the results can be seen above. All of this went according to plan and had no hang-ups of any kind. From the picture you can see that 3 of vectors took up inserts. The process of making the blots will be discussed in the next lab.