UIC Lab Blog

Rishika Gupta Internship Mankin Lab

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What I did

First I talked to Nora about starting the project.

Used plasmid pCS26-Pac as the cassette, it has luxC in it so the gene that allows E. Coli to produce light.

Kanomycene is the antibiotic that the plasmid has resistance to. 

I made competent cells, for both luxC and ermD.

I then isolated luxC plasmid.

I then isolated ermD plasmid.

I used the process of PCR to make more of the luxC gene.

I used the process of PCR makes more of the ermD.

I digested the plasmid to get rid of the background because I could not use fusion but I used Accu-prime instead.

We were going to do Gibson cloning so that we can stick the two genes that normally would not want to be together. sites are BanHI and BsrGI.

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The plasmid that I am making specifically has the ermC gene that will be expressed in the presence of ampicillian. 

Add more, based on Professor mankin's notes and stuff and then talk about everything youve been doing.

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The experiment that I'm doing could make work in the lab easier for researchers. Normally when you put your own plasmid into a bacteria you have to plate them on an agar plate with an antibiotic. If the bacteria has your plasmid it will show a resistance to that antibiotic and grow on the plate. If my experiment is successful we can use the plasmids that contain the gene in question of being expressed, and a gene that produces light when expressed. Researchers could use the plasmid that I created with a little part that can be removed and put another gene into it. This means it won't take having to plate the cells onto agar plates, but we would be able to test and see how much light is coming out of the cells. This would save a lot of time for researchers in the future because they could just add the gene they want to test to see if it will be expressed into my plasmid and then measure the amount of light that is coming from the cells. 

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PCR Reaction

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Cloning Vectors

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Preparing 1% Agarose Gel

We use agarose Gel to run plasmids and DNA fragments to test if reactions work. If we have a successful reaction the DNA band will be present and based on where it is we can determine if it is the correct size of the fragment that we originally wanted. It does not take that much DNA to run on an agarose gel so it is a very useful technique for making sure reactions worked properly.

Use 1 g of agarose for every 100mL of ethidium bromide. ex: If you want to make 250 mL of agarose gel measure out 2.5 g of agarose into 250mL ethidium bromide.

Combine agarose and ethidium bromide into an autoclaved jar. Remember to fill the jar a maximum of half way so that it doesn't boil over.

Microwave the jar until solution is clear. Remember to leave the cap ajar so the steam can escape the jar while boiling. 

Pour the gel into the plate, with the two stoppers at the ends. Add two combs to the gel and make sure the wells are not at the bottom of the plate but a little above it. Ethidium bromide is very toxic so be careful, pour the Agarose under the hood so you don't have to breathe the fumes. 

Let it sit 30-45 minutes before running DNA on the gel. 

Prepare the plasmids that you are running on the gel. Low concentration plasmids require 5uL of pladmid and 3uL of loading dye. High concentration plasmids require 2.5uL of plasmid and 3uL of loading dye. 

Also prepare the DNA Ladder to run beside your DNA fragments/plasmids.

when loading gels with two rows of wells, load the DNA onto the LOWER wells first. If you do not then the higher fragments will messy-up the lower wells so you can't distinguish the older DNA fragments from the newer ones.

When running the gel attach the red side to the bottom and the black side of the cable to the top. The negatively charged DNA will run from the wells toward the red (positive) side. 

Run the gel at 100-110 amps. for 15-20 min. Be careful to watch your gel, make sure the sample does not run out of the gel. 

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Preparing Glucose Stock

Glucose stocks are important because you can save cells that already have a certain plasmid in them. Instead of having to transform and put the plasmid that you have into the cells again you can culture cells straight from the glucose solution.

First take the cells have been incubating in Lb agar.

Transfer 850uL of the culture to a a 150mL eppendorf tube. 

Then add 150uL of 100% glycerol. The glycerol is very viscous so be precise about your pipetting.

Prepare an ethanol-dry ice reaction to flash freeze the cells before putting them into the -20C freezer. 

Flash freeze them for 5-10 minutes.

Put in -20C freezer for later use. 

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Pouring Agar Plates and Plating Transformed Cells

Agar plates are used in the lab to grow bacteria in a safe and controlled environment. It's important to remember that the cells that we grow are E.Coli but they are not the virulent type. We only grow competent cells, competent cells are special cells that are very weak and can take in plasmids though transformation (see Standard Transformation Protocol).

Get or make LB agar.

Melt it in the microwave. Make sure the lid is kept ajar so the steam escapes from the jar as its melting. 

Keep heating the LB until all the Agar has dissolved, there are no chunks or lumps of LB still in the jar.  

Once the agar is completely melted get Erlenmeyer flasks and a 50mL pipette. 

Prepare approximately 25mL of LB agar per plate. Use a pipette and transfer the amount of LB needed. Remember to transfer the LB near the Bunsen burner to reduce the amount of potential contamination of the LB. 

Let the LB agar cool until warm. If the LB is too hot when you add the antibiotic then the antibiotic will not be as effective. If you wait too long, the LB will start to harden in the flask and it will become lumpy on the plate. 

Add a 1uL of antibiotic for every mL of LB agar. ex: If you prepared 50mL of LB agar, add 50uL of antibiotic. Swirl the LB agar in the flask to distribute the antibiotic throughout the agar.

Pour the LB agar onto the plates near the flame. Fill the plates only half full. Be careful not to pour the bubbles! 

Once the plates have been poured you can leave the plates near the flame with the lids half off to cool.

Leave near the flame until completely cooled. 

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Credit to: 

Promega Corporation

2800 Woods Hollow Road

Madison, WI 53711-5399 USA

www.promega.com

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Plasmid Isolations

After transforming cells (see Standard Transformation Protocol) and plating cells (see 

Start out with the whole cells that have been incubating. They will be in LB medium. 

Spin the cells in a centrifuge for 5 minutes at 4000 rpm. After 5 minutes the cells should be pelted, if they are not spin longer. 

Remove remaining liquid which is known as supernatant. Be careful not to touch the pellet of cells at the bottom.

Add 250uL of suspension buffer/RNase. Resuspend the cells in the RNase. RNase is a very dangerous chemical in the lab because it gets rid of the RNA in a cell. Be very careful about using the RNase in the lab.

Transfer the resuspended cells into a 250mL eppendof tube. 

Add 250 mL of Lysis Buffer. Lysis buffer will break the cells so the contents of the cells will be free-floating in the solution.

Let them sit at room temperature for 5 minutes before preceding. The solution should become less cloudy. 

Add 350uL of chilled binding buffer. 

Put them on ice for 5 minutes.

Centrifuge them for 10 minutes at 13 rpm.

There will be protein at the bottom of the eppendorf tube, only remove the supernatent (liquid).

Put the supernatent in the stop of the filter and spin for 1 minute.Discard the flowthrough.

Add 500uL of wash buffer I. Centrifuge for 1 minute. Discard flowthrough.

Don't add anything, centrifuge for 1 minute to dry filter.

Put dry filter in a new eppendorf tube.

Add 50uL of Elution Buffer. Centrifuge for 1 minute, remove and discard filter.

End Result: 50uL of plasmid.

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Making LB Agar

Find the LB Agar powder 

Use 16 grams of LB powder for ever 400 mL of water. To prepare 250 mL of Agar, use 10 grams of LB powder and 250 mL of water.

Combine LB power and water in a pyrex jar. Make sure the solution is half the jar's maximum volume.

Put the jar with lid ajar into a metal tray and then into the autoclave. Put color changing tape on the top to make sure the autoclave is working correctly and the jar is in fact sterile when it comes out.

Take the LB agar out of the autoclave when done. Remember, the jar will be hot!

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Introduction

Over the summer of 2012, I have had the fortune to be able to work at the UIC Center for Pharmaceutical Biotechnology. I have been doing a summer internship for the past two summers with Pulkit Gupta and Professor Nora Lasop-Vasquez. I this blog I will record my experiences, experiments and how-tos for further use and for science fair. 

This year (school year of 2012-2013) I plan to present my experiments in the district of Chicago region 1's science fair.

Last summer I was working on a science experiment that didn't quite work out. It didn't go quite the way everyone was expecting but I got a lot of great practice working in the lab and doing tasks that prepared me for this summer. I kept a detailed lab journal of all the things I did, however I will not be posting about the work I did last summer because it may be confusing. I will provide all the "how-to's" that I've learned working in the lab from both years.