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Bio-Chemistry Lab Assignment: SDS-PAGE Registration No: FA20-BSI-080 Submitted To: Mam Bushra Ijaz Submitted By: Baseerat Fatima

The document describes an SDS-PAGE lab assignment to separate and analyze protein samples. Key steps included: 1. Collecting cell culture samples and adding them to labeled tubes along with sample buffer. 2. Denaturing the proteins by heating the samples. 3. Loading the samples and protein ladder into the wells of a polyacrylamide gel and conducting electrophoresis to separate the proteins by size. 4. An image of the resulting gel showed separation of the protein bands.

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80 views5 pages

Bio-Chemistry Lab Assignment: SDS-PAGE Registration No: FA20-BSI-080 Submitted To: Mam Bushra Ijaz Submitted By: Baseerat Fatima

The document describes an SDS-PAGE lab assignment to separate and analyze protein samples. Key steps included: 1. Collecting cell culture samples and adding them to labeled tubes along with sample buffer. 2. Denaturing the proteins by heating the samples. 3. Loading the samples and protein ladder into the wells of a polyacrylamide gel and conducting electrophoresis to separate the proteins by size. 4. An image of the resulting gel showed separation of the protein bands.

Uploaded by

black blood
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Date: 6-june-2021

Bio-Chemistry Lab Assignment: SDS-PAGE


Registration No: FA20-BSI-080
Submitted To: Mam Bushra Ijaz
Submitted by: Baseerat Fatima

Perform the online lab activity on the following link:

Write down:

1. Materials
2. Procedure
3. Results (image of gel)
Materials:
Reagents:
Chemicals that can be used in this experiment
1. 4x SDS Sample Buffer Solution
2. Wild Type Cell Culture
3. Pancreas Cell Culture
4. Edit Cell Culture
5. Edit 7 Cell Culture
6. Protein Ladder Solution
7. 1x Running Buffer

Micro pipetting Equipment:


Used to measure small volumes.
1. P2 Pipette
2. P20 Pipette
3. P200 Pipette
4. P1000 Pipette
5. P2 Tip Box
6. P20 Tip Box
7. P200 Tip Box
8. P1000 Tip Box
Empty Tubes:
Used to hold solutions.
1. Wild Type Cells Tube
2. Pancreas Tube
3. Edit 4 Tube
4. Edit 7 Tube

Electrophoresis Equipment:
Used to sort molecules by size and charge.
1. Polyacrylamide Gel
2. Protein Gel Electrophoresis Box
3. Power Supply

Other Equipment:
1. Microcentrifuge
2. Biohazardous Waste
3. Heat Block

Procedure:
1. Collect cells.
✓ Set the heat block's temperature to 95°C.
✓ Set the micropipette to 30µl on the P200.
✓ Attach a P200 tip.
✓ Place the tip of the micropipette in the wild type cell culture.
✓ By squeezing the plunger to the first stop, immersing the tip in the solution, and slowly
releasing the plunger, you can make a wild type cell culture.
✓ Close the cell culture of wild type cells.
✓ Place the tip of the micropipette in the wild type cells tube.
✓ Hold the plunger down until it reaches the second stop to dispense the wild type cell
culture into the wild type cells tube.
✓ Dump the tip into the trash with the eject button.
✓ To dispense 30µl of pancreas cell culture into the pancreas cells tube, repeat the
procedure.
✓ Repeat the technique to fill the edit 4 tube with 30 µl of edit 4 cell cultivations.
✓
✓ To distribute 30µl of edit 7 cell culture into the edit 7 tube, repeat the procedure.
2. Add sample buffer.
✓ Set the P20 micropipette to a volume of 10 ml.
✓ A P20 tip should be attached to a P20 micropipette.
✓ Place the tip of the micropipette in the 4X SDS sample buffer.
✓ By pressing the plunger to the first stop, immersing the tip in the solution, and slowly
releasing the plunger, 10µl of 4X SDS sample buffer can be made.
✓ Close the sample buffer for 4x SDS.
✓ Place the tip of the micropipette in the wild type cells tube.
✓ Hold the plunger down until it reaches the second stop to dispense the 4X SDS sample
buffer into the wild type cells tube.
✓ Toss the tip into the trash with the eject button.
✓ To distribute 10µl of 4X SDS sample buffer into the pancreas cells tube, repeat the process.
✓ To dispense 10µl of 4X SDS sample buffer into the edit 4 tube, repeat the procedure.
✓ To dispense 10µl of 4X SDS sample buffer into the edit 7 tube, repeat the procedure. SDS
sample buffer is already present in the protein ladder.

3. Denature the proteins.


✓ Place all four sample tubes on top of the heat block.
✓ Set the timer on the heat block to 10 minutes and push the start button.
✓ Remove the sample tubes from the heat block one by one.

4. Centrifuge the samples.

✓ Open the lid of the microcentrifuge and place your four sample tubes inside. It is not
necessary to spin down the protein ladder.
✓ Close the microcentrifuge lid and arrange the 4 tubes so that the filled receptacles are
symmetrical.
✓ To quickly spin the liquid to the bottom of the solution tubes, press the pulse button.

5. Load the gel.

✓ Take the tubes out of the microcentrifuge on electrophoresis box and set them aside.
✓ Fill the gel cassette with 1X Running buffer until the wells of the gel are completely
covered.
✓ Take the comb out of the gel cassette.
✓ 1X running buffer should be added to the outer gel electrophoresis tank.
✓ Set the P200 micropipette to a volume of 20µl.
✓ Add a P200 tip to the mix.
✓ Place the tip of the micropipette in the protein ladder tube.
✓ By pressing the plunger to the first stop, immersing the tip in the solution, and gently
releasing the plunger, you can create a protein ladder.
✓ Place the tip of the micropipette above the first lane of the protein gel.
✓ Hold the plunger down until it reaches the first stop to dispense the protein ladder into
the first well.
✓ Toss the tip into the trash with the eject button.
✓ In the second well of the gel, repeat the procedure to dispense 20 µl of the positive
control (wild type cells).
✓ Repeat the procedure to fill the third well of the gel with 20 µl of the negative control
(pancreas cells).
✓ Repeat the procedure to pour 20 l of edit 4 cells into the gel's fourth well.
✓ Repeat the procedure to dispense 20µof edit 7 cells into the gel's fifth well.

6. Conduct protein electrophoresis.

✓ Cover the electrophoresis box with the cover.


✓ Connect the red and black cables to the red and black connection points on the power
supply, as well as the red and black connection points on the gel electrophoresis box's lid.
✓ Activate the power supply.
✓ Set the power supply at 150 volts.
✓ set the timer for 60 minutes.
✓ The power supply should be turned on.
✓ Wait until the timer goes off.
✓ Turn off the power switch .
✓ Disconnect the electrodes from the electrical supply.
✓ To inspect your gel, carefully remove the cover from the electrophoresis box.

Results(Image of Gel):

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