Podcast
Questions and Answers
What should you do if the micropipette's volume knobs are forced past their limits?
What should you do if the micropipette's volume knobs are forced past their limits?
- This is the correct procedure for calibration.
- It is safe to adjust them as needed.
- No action is necessary; they are designed for this.
- You might damage the micropipette. (correct)
What is the first step to using a micropipette for accurate measurement?
What is the first step to using a micropipette for accurate measurement?
- Start distributing the liquid immediately.
- Choose a tip that is too large to ensure a good fit.
- Always hold the micropipette upside down.
- Adjust the volume to the desired amount. (correct)
How should you check if you have pipetted the correct volume into the 1.5 mL tube?
How should you check if you have pipetted the correct volume into the 1.5 mL tube?
- Weigh the tube and its contents.
- Confirm with another student.
- Self-check using the graduation marks on the tube. (correct)
- Estimate visually by looking at the tube.
Which of the following is NOT a correct practice when using a micropipette?
Which of the following is NOT a correct practice when using a micropipette?
What volume should you practice pipetting immediately after confirming comfort with the P-1000?
What volume should you practice pipetting immediately after confirming comfort with the P-1000?
What happens after you press the plunger to the first stop and immerse the tip into the solution?
What happens after you press the plunger to the first stop and immerse the tip into the solution?
What should you do with the used 1.5 mL tube after completing the exercise?
What should you do with the used 1.5 mL tube after completing the exercise?
Which step is performed after obtaining a new 1.5 mL microcentrifuge tube?
Which step is performed after obtaining a new 1.5 mL microcentrifuge tube?
What should be labeled on the lids of the 1.5 mL microcentrifuge tubes?
What should be labeled on the lids of the 1.5 mL microcentrifuge tubes?
What is the first step to follow when preparing the reagents for Tube A?
What is the first step to follow when preparing the reagents for Tube A?
After mixing the contents of Tube A, what should you do next?
After mixing the contents of Tube A, what should you do next?
What does a properly pipetted sample in Tube A indicate?
What does a properly pipetted sample in Tube A indicate?
How should the accuracy of pipetting be double-checked for Tubes B and C?
How should the accuracy of pipetting be double-checked for Tubes B and C?
What should happen to the 1.5 mL tubes after they have been used?
What should happen to the 1.5 mL tubes after they have been used?
What is the total volume of solutions combined in Tube B?
What is the total volume of solutions combined in Tube B?
Which of the following is a common use of absorbance spectrophotometry in laboratory procedures?
Which of the following is a common use of absorbance spectrophotometry in laboratory procedures?
What should be considered to avoid introducing air bubbles while pipetting?
What should be considered to avoid introducing air bubbles while pipetting?
What is the optimal method for dispensing the solution from a pipette tip?
What is the optimal method for dispensing the solution from a pipette tip?
What should be done with the pipette tip after pipetting different solutions?
What should be done with the pipette tip after pipetting different solutions?
When mixing reagents in a 1.5 mL tube, which reagent should be added first?
When mixing reagents in a 1.5 mL tube, which reagent should be added first?
What technique is recommended for mixing reagents in a microcentrifuge tube?
What technique is recommended for mixing reagents in a microcentrifuge tube?
What is the purpose of centrifuging reagents in a microcentrifuge tube after mixing?
What is the purpose of centrifuging reagents in a microcentrifuge tube after mixing?
Which statement reflects a common misconception regarding pipetting accuracy?
Which statement reflects a common misconception regarding pipetting accuracy?
How should the end of the pipette tip be positioned when inserting it into a solution?
How should the end of the pipette tip be positioned when inserting it into a solution?
What is the relation of OD260 of 1 for double-stranded DNA in terms of concentration?
What is the relation of OD260 of 1 for double-stranded DNA in terms of concentration?
What is the OD260/OD280 ratio indicative of a pure dsDNA solution?
What is the OD260/OD280 ratio indicative of a pure dsDNA solution?
Which wavelength is primarily used to assess the concentration of nucleic acids?
Which wavelength is primarily used to assess the concentration of nucleic acids?
If the OD260 of a dsDNA sample is 0.70, what is the concentration of the sample?
If the OD260 of a dsDNA sample is 0.70, what is the concentration of the sample?
What does an OD260/OD280 ratio smaller than 1.8 generally suggest?
What does an OD260/OD280 ratio smaller than 1.8 generally suggest?
What is the result of dividing OD260 of 0.70 by OD280 of 0.39 for a dsDNA solution?
What is the result of dividing OD260 of 0.70 by OD280 of 0.39 for a dsDNA solution?
Which measurement can indicate contamination by urea in nucleic acid solutions?
Which measurement can indicate contamination by urea in nucleic acid solutions?
What is the purpose of serially diluting a DNA solution before analysis?
What is the purpose of serially diluting a DNA solution before analysis?
What is the purpose of the 'Touch' mode on the benchtop vortex?
What is the purpose of the 'Touch' mode on the benchtop vortex?
Which dial is used to control the duration of centrifugation in a microcentrifuge?
Which dial is used to control the duration of centrifugation in a microcentrifuge?
What should be done after placing samples in the rotor of a microcentrifuge?
What should be done after placing samples in the rotor of a microcentrifuge?
How does the rpm/rcf dial affect the operation of a microcentrifuge?
How does the rpm/rcf dial affect the operation of a microcentrifuge?
What is the maximum speed behavior of the microcentrifuge when the 'short' button is held?
What is the maximum speed behavior of the microcentrifuge when the 'short' button is held?
What does the term 'rcf' refer to in the context of centrifugation?
What does the term 'rcf' refer to in the context of centrifugation?
Why is it important to operate a microcentrifuge with balanced samples?
Why is it important to operate a microcentrifuge with balanced samples?
What should be done if unusual noises or shaking are heard during operation?
What should be done if unusual noises or shaking are heard during operation?
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Study Notes
Micropipetting Technique
- Micropipettes have a plunger with two stops; the first provides gentle resistance, and the second dispenses the full volume.
- Practice using the micropipette with colored water in 1.5 mL microcentrifuge tubes before performing experiments.
- Confirm pipetting accuracy with graduation marks on the tube and get TA approval before proceeding.
Micropipette Usage Guidelines
- Use micropipettes carefully, as they are delicate and costly to replace.
- Always select the appropriate micropipette based on the volume you are handling, and adjust the knob within its limits.
- Attach tips without force; immerse the tip in the solution only after pressing to the first stop.
- Release the plunger slowly to avoid introducing air bubbles.
- To dispense, gently press the plunger to the first stop, followed by the second stop for complete solution release.
Reagent Mixing Procedure
- For setting up reactions, always add the largest volume reagent first in the 1.5 mL microcentrifuge tubes.
- Mix reagents by vortexing or flicking the tube after all components are added.
- Centrifuge briefly to ensure all liquid is gathered at the bottom of the tube, avoiding reagent loss.
Spectrophotometric Analysis of Nucleic Acids
- Use spectrophotometry (OD260) to measure nucleic acid concentration; for dsDNA, 1 OD260 equals 50 µg/mL, while for ssDNA or RNA, it equals 40 µg/mL.
- Assess sample purity using OD260/OD280 ratios—values of 1.8 for dsDNA and 2.0 for RNA indicate purity; lower ratios suggest contamination.
- Perform serial dilution if the nucleic acid concentration is too high for direct measurement, targeting a final dilution of 200x.
Nanodrop Spectrophotometer Operation
- Use a Nanodrop to analyze DNA solutions efficiently with smaller sample volumes.
- The device calculates concentration and purity of nucleic acids but requires manual verification of calculations.
- When operating, ensure the selector is on 'ON' for continuous motion and 'Touch' for vortex mode.
Microcentrifuge Operations
- Use benchtop microcentrifuges for 1.5 mL tube centrifugation to pellet particles and collect solutions.
- Load samples in a balanced configuration and secure with a rotor cover.
- Control speed (RPM or RCF) and duration of centrifugation to optimize results.
- Always monitor for unusual noises or movement during operation and stop the centrifuge if necessary.
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