Biology 475

Biology 475

Molecular Biology

Lab Two – Library Screening With

Restriction Enzymes

Kelly L. Shipley

Introduction

Now that our samples of 16s genes have been cloned, we will use our clone library to screen for inserts. Restriction enzymes, EcoRI, will cut the plasmid at the EcoRI sites, releasing the insert from the plasmid if it is present. Gel electrophoresis using our digest DNA samples will verify the presence of vector alone, or vector plus insert.

Cloning Results

Results: Our library of cloned E. coli colonies contains many possible variants of the 16s gene, which may or may not contain our insert. Using genetic markers, such as lac Z and antibiotic resistance gene ampicillin, we will test to see whether the insert is present in the bacterial cell. Amp is included in the nutrient within this Petri dish, only E. coli which has taken up the vector will be able to grow, so our library contains only E. coli which has taken up the AntR vector, and at this point we are unsure of the presence of the insert.

Mini-Prep Methods

Methods: E. coli from our library has been added to nutrient broth with ampicillin to aid in the construct. Our samples are now ready for the rapid boiling method of isolating the plasmid from the bacterial cell components.

Methods: After pelleting and resuspending with STET, lysozyme is added to each test tube to break open the E. coli cell. After this step, boiling, centrifuging and removal of the pellet leaves supernatant containing plasmids only.

	Restriction Methods and Results
Digestion Methods: Test tubes containing our DNA samples, buffer, EcoRI and water are placed in a floating rack within a water bath to incubate at 37 degrees C for 45-90 minutes. The purpose of the water bath is to give the EcoRI enzymes the optimal environment for cutting the plasmid if the insert is present.
Electrophoresis Methods: A 1% agarose gel is used in our gel apparatus. The gel is loaded from left to right, Lane 1 is loaded with the standard DNA marker, Lane 2-11 are loaded with our digest results containing plasmid only or plasmid plus insert and Lane 12 is empty.
Results: Molecules in the gel migrate according to size and charge. After cutting the plasmid with EcoRI, loading our samples into the gel and letting it run, our results clearly show the molecular standard in Lane 1 (far left), Lanes 2,3,4,6,7,8,11 showing bands correlating to the size for both vector and insert, while Lanes 5,9 and 10 show only bands corresponding with vector. Lane 1- DNA Marker Lane 2- Sample #51 Lane 3- Sample #52 Lane 4- Sample #53 Lane 5- Sample #54 Lane 6- Sample #55 Lane 7- Sample #56 Lane 8- Sample #63 Lane 9- Sample #64 Lane 10- Sample #65 Lane 11- Sample #66 Lane 12- Blank	Lanes 1 through 12

Discussion

Using the rapid-boiling method for plasmid isolation, we have begun to prepare our sample for eventual use in DNA sequencing analysis. Our sample has been added into solution with plasmids ready to accept an insert of DNA, and then mixed with E. coli. Plasmids purchased have an antibiotic resistance gene to ampicillin and the lac Z gene, both of which help us determine the presence of the plasmid and the presence of the insert respectively. After our clone library is complete, samples are taken for testing to positively determine the presence of the insert, our target DNA.

In this lab we used restriction enzyme, EcoRI, to cut our plasmid at the EcoRI sites. To determine whether the EcoRI did this and if the target DNA insert was present, we used gel electrophoresis. Our results show that the insert and plasmid were present in samples 51, 52, 53, 55, 56, 63 and 66. These sample numbers correlate to the clone colonies of the same numbers from the clone library.

From this we know our target DNA was indeed taken up by the plasmid within the E.coli during heat shocking of the samples. This positive identification of the 16s rRNA gene target gives us an individualized colony for future use in DNA sequence analysis.