# data[].attributes.body
Once the GFP gene is produced, scientists can attach tags to selected proteins to monitor cellular activity in organisms.

# data[].attributes.body
The GFP is made from a gene that is specifically designed to create GFP.

# data[].attributes.body
This GFP from the jellyfish is able to be produced as a result of DNA Recombinant Technology.

# data[].attributes.body
GFP is also related to insulin because insulin can be produced from the transcription of the bacterial cell´s plasmid.

# data[].attributes.body
Agitate on a rocking platform for 2-3 hours.

# data[].attributes.body
Allow the Protein InstaStain paper to stain the gel for about an hour at room temperature.

# data[].attributes.body
Gently float a sheet of Protein InstaStain with the stain side (blue side) down in the staining/destaining solution. Cover the gel with plastic wrap to prevent evaporation.

# data[].attributes.body
Carefully remove the back plate from the tray.

# data[].attributes.body
Add a sufficient volume (approximately 100 ml) of the staining/destaining solution into the tray to cover the gel and back plate.

# data[].attributes.body
After gel electrophoresis has been performed, lay the cassette down and remove the front plate.

# data[].attributes.body
Perform gel electrophoresis on the GFP protein so that the GFP samples can be analyzed.

# data[].attributes.body
Rinse each well by squirting electrophoresis buffer into the wells using a transfer pipet. Once this step is done, the gel will now by ready for GFP protein sample loading because the buffer will now allow the GFP´s molecules to be separated by charge.

# data[].attributes.body
Add diluted electrophoresis buffer to the chamber.  The purpose of adding the buffer is transmit charge throughout the gel.

# data[].attributes.body
Insert the gel into the electrophoresis chamber. The gel will promote the separation of the GFP protein´s molecules.

# data[].attributes.body
Carefully remove the comb by gently pulling upwards. Pull the comb straight up to prevent damage to the wells.

# data[].attributes.body
Gels may feature a sticker or tape at the bottom of the front plate. Remove the tape if present so that the bottom of the gel will be exposed.

# data[].attributes.body
Boil the GFP sample for 5 minutes. Afterwards, remove the sample tube from the beaker and allow it to cool for a few minutes at room temperature.

# data[].attributes.body
Push the bottom of the GFP sample tube through the foil and immerse in the boiling water. The protein in the protein denaturing solution, along with the boiling water, will help denature the GFP protein.

# data[].attributes.body
The purpose of denaturing proteins is to prevent the loss of the protein´s function (in this case, the GFP protein). To denature the protein sample, add 10 micro liters of protein denaturing solution to the tube labeled ¨GFP denatured¨ and mix well. The denaturing solution contains sodium dodecyl sulfate (SDS) and 2-mercaptoethanol.

# data[].attributes.body
Click to see image.

# data[].attributes.body
Click to see image.

# data[].attributes.body
Click to see image.

# data[].attributes.body
Click to see image.

# data[].attributes.body
The purified GFP Protein will look like the image below.

# data[].attributes.body
Transfer an additional 30 micro liters of the same elution to a second screw-top microcentrifuge tube. This tube will be labeled ¨GFP Denatured.¨

# data[].attributes.body
Transfer 30 micro liters of the brightest elution to a screw-top microcentrifuge tube. This tube will be labeled ¨GFP Native.¨

# data[].attributes.body
When the GFP Protein band almost reaches the bottom of the column, start collecting the fractions in the microtiter plate. In order to prevent any loss of GFP protein, begin collecting fractions before the GFP reaches the bottom of the column.

# data[].attributes.body
By using GFP as an imaging source, if the GFP test shows that the pancreas is not making enough insulin for the cells, doctors can help better diagnose and treat diabetic patients.

# data[].attributes.body
GFP is related to diabetes and insulin because the protein acts as an imaging source to determine whether or not enough insulin is being secreted by the pancreas and into the cells.

# data[].attributes.body
Once these steps are accomplished, the bacterial cells should be lysed like the cells in the microscopic diagram.

# data[].attributes.body
Remove the cap so that the extract can completely enter the column, collecting the ¨flow-through¨ waste into the beaker.

# data[].attributes.body
Begin to elute the column with 1X elution buffer. Add the buffer slowly. The purpose of eluting the column with 1X elution buffer is to avoid diluting the protein sample.

# data[].attributes.body
Slowly load the column with 0.2 ml of the GFP extract.

# data[].attributes.body
Wash the packed column with 5 ml of 1X elution buffer. Always keep a thin layer of elution buffer on top of the packed matrix to prevent the solution from drying.

# data[].attributes.body
Once the beaker is placed underneath the column, remove the cap from the bottom of the column so that the matrix can be packed into the column

# data[].attributes.body
Place an empty beaker under the column in order to collect the 1X elution buffer.

# data[].attributes.body
Pipet 4 ml of the mixed slurry into the column by letting it stream down the inside walls of the column. The purpose of adding slurry is to have more efficiency in separating the green fluorescent protein.

# data[].attributes.body
Add 1 ml of 1X elution buffer to the column to prevent the column from drying.

# data[].attributes.body
Vertically mount the column on a ring stand. Make sure it is straight and that the white cap is firmly attached to the bottom of the column.

# data[].attributes.body
Click to see image.

# data[].attributes.body
Click to see image.

# data[].attributes.body
Centrifuge the tube in a microcentrifuge for 5 minutes to further lyse the cells.

# data[].attributes.body
Repeat steps 9-12 two more times. The purpose of the freezing, melting, and vortexing pattern is to help lyse the bacterial cells.

# data[].attributes.body
Vortex the samples vigorously for 30 seconds.

# data[].attributes.body
After the cell suspension is completely frozen, remove the centrifuge tube from the freezer and put it in a 55 degree Celsius water bath to thaw the cells.

# data[].attributes.body
Place the microcentrifuge tube containing the GFP cells in a -20 degree Celsius freezer for 15 minutes.

# data[].attributes.body
Incubate tube for 10 minutes in a 55 degree Celsius bath.

# data[].attributes.body
Vortex the bacterial tube at maximum speed. The purpose of vortexing is to make sure the cells are fully resuspended.

# data[].attributes.body
Twirl the loop containing the colonies in the tube containing lysis buffer. The purpose of twirling is to dislodge the cells into the buffer solution.

# data[].attributes.body
Select a GFP plate showing the highest GFP expression (maximum glow). Using a sterile loop, scrape the cell growth off of the GFP plate to further isolate the cells.

# data[].attributes.body
Incubate the plates in a 37 degree incubator overnight.

# data[].attributes.body
Repeat the same process as steps 1 and 2 for the second plate.

# data[].attributes.body
Spread the cells evenly and thoroughly over the entire surface of the plate to allow for even distribution of the cells. Turn the plate 90 degrees and thoroughly spread again using the same loop.

# data[].attributes.body
With an inoculating loop, pick 4-5 isolated GFP expressing (glowing) colonies to isolate the colonies that have the GFP Protein.

# data[].attributes.body
Click to see image.

# data[].attributes.body
Click to see image.

# data[].attributes.body
After performing gel electrophoresis on the GFP protein samples, the results should look like this picture.

# data[].attributes.body
After performing these steps, the transformed bacteria should look like the bacteria on the right side of this image.

# data[].attributes.body
GFP was originally discovered from the Aequorea Victoria species of jellyfish.

# data[].attributes.body
Spread the cells over the entire plate using an inoculating loop. Use one sterile loop to spread both -DNA samples. Change to a fresh loop before spreading the +DNA samples. The cells are being spread over the plates so that the cell suspensions will be absorbed by the agar solution and to promote the bacteria to grow.

# data[].attributes.body
Using a new sterile 1 ml pipet, transfer 250 micro liters of recovered cells from the tube labeled ¨+DNA¨ to the middle of the +DNA/+Amp and +DNA/+Amp plates.

# data[].attributes.headline
2a. and 2b.) Three Main Parts and Why Each was Important

# data[].attributes.subject
2a. and 2b.) Three Main Parts and Why Each was Important

# data[].attributes.headline
1c.) How does GFP Relate to Insulin and Diabetes

# data[].attributes.subject
1c.) How does GFP Relate to Insulin and Diabetes

# data[].attributes.headline
1b.) Explain how GFP is Created and what is Needed to Make it Happen

# data[].attributes.subject
1b.) Explain how GFP is Created and what is Needed to Make it Happen

# data[].attributes.body
Using a sterile 1 ml pipet, transfer 250 micro liters of recovered cells from the tube labeled ¨-DNA¨ to the middle of the -DNA and
-DNA and -DNA/+Amp plates.

# data[].attributes.body
Purpose: The purpose of isolating the GFP protein is so that the GFP can be separated from other materials, such as molecules or other proteins.

# data[].attributes.headline
Module 1 Procedure Diagram (Continued) (Click on Picture for Detailed View)

# data[].attributes.subject
Module 1 Procedure Diagram (Continued) (Click on Picture for Detailed View)

# data[].attributes.headline
Module 1 Procedure Diagram (Click on Picture for Detailed View)

# data[].attributes.subject
Module 1 Procedure Diagram (Click on Picture for Detailed View)

# data[].attributes.attachment_link.title
Protein Lab (Module 1 Procedure Diagram)

# data[].attributes.body
The techniques that are required to create a pFlurogreen plasmid are...

# data[].attributes.headline
ii. Techniques Required to Create the Plasmid

# data[].attributes.subject
ii. Techniques Required to Create the Plasmid

# data[].attributes.body
Once recovery broth has been added to the bacterial tubes, incubate the cells in a 37 deg C water bath for 10 minutes to continue to allow the plasmid DNA to interact with the bacteria.

# data[].attributes.body
Once the bacterial cells have incubated on the ice for 2 minutes, 250 micro liters of recovery broth must be transferred in order to promote improved cell viability and greater cloning efficiency.

# data[].attributes.body
After the bacteria has been in the water bath for 45 seconds, place the bacteria back on the ice for 2 minutes so that the bacteria can continue to incubate and interact with the calcium chloride.

# data[].attributes.body
The main functions of the GFP Protein are to tag proteins and to examine cellular processes within living cells.

# data[].attributes.body
araC is another important part of the pFluorogreen Plasmid, which is in charge of the expression of the GFP Protein.

# data[].attributes.body
Bla (beta-lactamase enzyme) allows for bacteria to become resistant to the beta-lactam class of antibiotics (penicillin class of antibiotics).

# data[].attributes.body
The main structures of a pFluorogreen Plasmid include the following genes.

# data[].attributes.body
Once the bacteria has incubated on the ice for 10 minutes, place the transformation tubes in a 42 deg C water bath for 45 seconds. The purpose of putting the bacteria colonies in the water bath is to allow the plasmid DNA to interact with the bacteria.

# data[].attributes.body
Incubate the tubes on ice for 10 minutes so that bacteria can interact with the calcium ion.

# data[].attributes.body
Transfer 250 micro liters of the cell suspension to the ¨+DNA¨ tube and place both tubes on ice.

# data[].attributes.headline
Step #2 (Refer to Slide #3 for Image of the Process)

# data[].attributes.subject
Step #2 (Refer to Slide #3 for Image of the Process)

# data[].attributes.body
The next part of the transformation process is to twist an inoculating loop to free all the cells and make sure each cell is transformed with the GFP Protein.

# data[].attributes.body
The first step in the transformation process is to add calcium chloride (CaCl2) to the bacteria cell. This will allow for the bacteria to incorporate it´s plasmid DNA and allow for an easy transformation process.

# data[].attributes.body
Purpose:  The reason why protein gel electrophoresis is being used in this lab is so that the GFP samples (both the native and denatured) can be analyzed.

# data[].attributes.body
Purpose: The purpose of protein purification is to separate the protein of interest from other materials, such as cell particles or other proteins.  This process is vital for the characterization of the function, structure, and interactions for the desired protein (in this lab, the GFP protein).

# data[].attributes.body
Purpose: The purpose of bacterial transformation is to introduce a foreign plasmid into bacteria.  The bacteria can then amplify the plasmid and make large quantities of it.

# data[].attributes.body
The overall purpose of the Protein Factories lab is to transform and purify Green Fluorescent Protein (GFP) in bacteria.