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Experiment # 1
Title: Pipettes and electrophoresis
(agarose and acrylamide gels)
Purpose:
- The goal of this experiment is to identify and be able to use different types of pipettes and to conclude that which pipette is more efficient. We should be able to differentiate between accuracy and precision. The purpose is to learn how to measure volume in microlitres using pipettes.
- We should have knowledge about electrophoresis process and its types.
Procedure:
Part 1:
- Took a graduated glass pipette and an automated pipette.
- Graduated volume pipette was set at 1100 μL and variable volume pipette at 1000 μL.
- Took an empty beaker and placed it on weighing balance and tared it to zero.
- Took graduated glass pipette, wet the thumb and placed it on the end of pipette that is not used in solution.
- Placed bulb on that side of pipette, dipped the other end in water and extracted water by squeezing the bulb until it reached the measurement value.
- Then released the measured water in the beaker at weighing balance. When the required volume reached, stopped the water from coming out by placing the thumb over the pipette.
- Measured the weight and noted it.
- Repeated the procedure four more times.
- Took the automated pipette. Twisted the knob on the top of the pipette to set the value of pipette.
- Dipped the tip in water. Pushed the button at top and released the button to slowly fill the pipette.
- Stop 2 in pipette is to dispense the solution. To release most of the water in pipette, pushed down to first stop. Then stop 2 released the rest of water in beaker.
- Noted the weigh of measured volume of water. Tared the weighing balance and repeated the process four more times.
- Stop 3 was used to eject the tip and discarded it.
- To measure accuracy, set the pipettes at different volumes from 100 -1000 μL. Repeated the procedure for each volume and calculated the difference of measured volume from set volume.
Part 2:
SDS-Page electrophoresis:
- Took SDS-Page gel and removed clip and tape from bottom.
- Placed it into gel holder in such a way that its lower part goes to interior of cassette.
- To make sure that there are no leaks, added buffer solution.
- Filled it with buffer solution until it is above the lower plate and placed gel holder in gel tank.
- Prepared the proteins by boiling them in water in presence of denaturing buffer for about 5-10 min. Then put them into ice water and cooled them.
- Loaded 10 microlitre of molecular weight marker in pipette and placed into well 2.
- Placed 20 μL of Dog 2 (Sparky) sample in lane 5.
- Placed 20 μL of Dog 3 (Scooby) sample in lane 6.
- Closed the lid and switched on the power source. Set the power source at 200 volt and left it to run for 30-40 minutes.
- Poured out the buffer, removed the gel from tank and analysed the results.
Data / calculations / results:
Part 1:
Pipette precision: (Graduated vs automated pipette)
Converted all masses of water into volume by dividing mass with density of water at 22 ℃, which is 0.99777 g/mL. Measured every value and divided it by the total number of values to determine the mean value. To compute the standard deviation, take the mean of each measurement and square the result. Divide the total of those values by the number of measurements minus one. determined the mean and standard deviation using the formula: Variable volume pipette Graduated pipette Trial Mass (g) Volume (mL) Trial Mass (g) Volume (mL) 1 1.0482 1.0505 1 1.0089 1. 2 1.0075^ 1.0098 2 0.9904 0. 3 0.9914 0.9936 3 0.9951 0. 4 0.9908^ 0.9930 4 1.0054 1. 5 0.9853^ 0.9875 5 0.9863 0.
Mean 1.0069 Mean 0. Standard deviation 0.0260 Standard deviation
Pipette accuracy (variable volume vs graduated glass pipette):
Part 2:
SDS-Page electrophoresis
Rf = x/y Y = 5.4cm, maximum distance covered by dye Rf^ Log 10 MW
Unknown sample DNA matches with DNA sample of sparky.
Discussion questions:
Q #1: How is a glass pipette similar and different from an automatic pipettor? Liquids are transferred and volume is measured using both automated and glass pipettes. Both of them are used to measure volume accurately and with minimal loss. However, they can have a specific volume limit and be graded or volumetric in glass pipettes. However, the capacity of the automatic pipette can be changed. An automated pipette has mechanical control, whereas a glass pipette requires manual adjustment and is slower to use. Glass pipettes must be cleaned after each use; however automated pipettes' tips can be thrown away after usage. A pipette that is automated is more accurate. Q#2: What is the difference between accuracy and precision? While precision refers to how calculated values are near to one another even when they are not close to the true value, accuracy refers to how close the value is to the actual value or desired value. While precision is about getting consistent outcomes, even if they are not precise, accuracy is about getting the right value. Q#3: When pipetting how should you treat volatile organic compounds differently than water-based solutions?
Organic liquids continuously fall out of the pipette when being pipetted out. The liquid is repeatedly drawn into the pipette to stop this phenomenon. The pressure is balanced, and the organic molecule stops leaking out. Water based solution do not drip out of pipette. Q#4: Is electrophoresis an analytical or preparative technique? Explain giving one piece of evidence in your rationale. Usually, electrophoresis is employed as an analytical method. For instance, agarose gel electrophoresis is used to analyze and visualize DNA material. It separates molecules according to their sizes.
Conclusion:
The automated pipette is more accurate and precise compared to manual pipette. Using different types of pipettes changes the outcome of experiments. We learned how to measure small amount of volume using pipettes. With electrophoresis technique, we can separate and analyse molecules based on their sizes. We can measure the molecular weight of unknown proteins using SDS-Page electrophoresis. And agarose gel electrophoresis can be used to identify DNA samples and can be used in crime scenes or investigations.
Notes:
Agarose is long chain of carbohydrates. Agarose electrophoresis is used to identify DNA in crime scenes. SDS-Page electrophoresis is used for proteins.
