Team:Toronto/Experiment-Nanodrop

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Nanodrop

Introduction

Nanodrop machines are spectrophotometric machines designed to quantitively measure the concentration of DNA or RNA present in the mixture Safety Precausions SDS (Safety data sheet): Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding the information on these sheets. PPE (Personal protective equipment): Proper lab attire should be worn throughout the experiment: This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back.

Materials

• Reagents
  • DNA sample
  • Diethylpyrocarbonate water (DEPC H₂O)
  • Ellution buffer (from kit)
• Equipment
  • Nanodrop machine
  • Micropipette
  • Centrifuge
  • Kimwipes

Procedure

• Preparation

1. Quick run centrifuge all of the liquid with your DNA sample in it until it falls to the bottom. (AZ: Why??? This will mess with your nanodrop readings)

2. Bring with you a P2 pipette and appropriate tips, elution buffer (or whatever your DNA is suspended in, for the blank), and DEPC water.

• Set up

3. Clean the Nanodrop with a Kimwipe before use, and after each sample has been loaded.

4. Turn on the software by clicking on the Nanodrop Icon on the computer

5. Select Nucleic Acid and Follow instructions given

• Taking Samples

6. Before making measurements, commant will prompt you to load a 2 μL sample of DEPC water in order to initialise the instrument. Raise the Nanodrop arm and pipette this volme onto the round silver pedestal closest to you, making sure that your elbows are placed firmly on the bench and both hands are steadying the pipette. Lower the arm of the Nanodrop and OK when this sample is loaded. (AZ: 0.5uL of sample should be enough for getting a good read) The rectangle in the bottom right corner should read: ng/μL (nanogram/microliter): NaN.

7. Repeat the loading procedure above for your elution medium: if the elution buffer from a Miniprep kit was used, pipette that onto the Nanodrop; however, if DEPC water was used to elute DNA, then another DEPC water sample must be loaded. Once this is completed. lower the arm and press Blank (N.B. NOT Re-Blank, which is a different commant).

• Before you Leave

• Place a kimwipe over the cleaned pedestal and lower the arm over it

• Troubleshooting

8. Always clean the pedestals after all samples are read and do a measurement on a fresh replicate of the blanking solution to confirm that the pedestal is clean.

9. The "Re-Blank" option establishes a new reference which is used for the calculations of all subsequent samples. This is useful if you start getting weird measurements.

• References

• Adam

• Operations Manual http://memphys.dk/sites/default/files/files/basic_page/%3Cem%3EEdit%20Basic%20page%3C/em%3E%20UV-Vis%20spectroscopy/nd­1000­v3.7­users­manual­8.5x11.pdf

• Retrieved from "http://local.biochemistry.utoronto.ca/igem/index.php?title=Nanodrop&oldid=995" Category: Lab Protocols

• Changelog

• 5/16/2016

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