NanoDrop Instructions

NanoDrop Instructions

Nanodrop Instructions

General info

• UV-Vis Spectrophotometer

• Only needs 1-2 µL sample

• Assesses quality and quantity

• Wireless data transfer- will it reach 5th floor from Nanodrop on 4th floor? Probably easiest to use USB flash drive

• View data using Nanodrop One/Oneᶜ Viewer software

Important instructions:

• Only wipe upper and lower pedestals with a clean, dry, lint-free wipe. Do NOT use a paper towel

• Do NOT spray or squirt any liquid onto the NanoDrop

• Only micropipettes should be used to dispense liquid onto the lower pedestal

• When measuring samples IMMEDIATELY click Measure after lowering the sampling arm

• Wipe pedestals clean ASAP after taking sample measurements so the sample doesn’t dry on the pedestals

• Use a fresh pipette tip and fresh drop of sample for each measurement

• Always clean the pedestals with dH₂O and a lint-free wipe at the very beginning and very end of taking measurements

When you go down to Dr. Chang’s lab on the 4th floor bring:

• Samples

• Micropipette

• Pipette tips

• Gloves

• Also don’t forget to introduce yourself and ask Dr. Chang if it’s okay to use the Nanodrop

Measuring DNA and RNA

1. Clean pedestal surfaces prior to starting

   • Cleaning

     • Use dH₂O and a lint-free laboratory wipe to clean upper and lower pedestals, and then dry with kimwipe

     • Do NOT spray or squirt water onto NanoDrop

     • If there is a dried sample stuck on the pedestals use a 3 µL drop of HCl followed by 3 µL of dH₂O to clean

   • Reconditioning (DO NOT HAVE TO DO)

     • Use the applicator in the reconditioning kit, take a pin-head sized amount of NanoDrop Pedestal Reconditioning Compound (PR-1)

     • Apply a thin even layer of PR-1 to the flat part of the lower and upper pedestals

     • Wait 30 seconds for the PR-1 to dry

     • Fold a lint-free laboratory wipe into quarters and wipe PR-1 off until no more dark PR-1 compound residue shows up on the wipe

2. Take a blank measurement using the same buffer as used in the sample

   • Pipette 1- 2 µL of blank buffer (usually MBG H₂O) onto lower pedestal

   • Lower sampling arm

   • Click Blank

   • Wipe pedestals with dry, lint-free wipe

   • Do NOT use samples or blank buffers containing hydrofluoric acid (HF)

   • dH₂O and TE are common blank buffers used for nucleic acids

3. Gently vortex sample

   • Note nucleic acid sample must be purified prior to measurement

4. Pipette 1-2 µL of the sample onto the lower measurement pedestal

   • 1 µL is usually sufficient but 2 µL is better if the sample has reduced surface tension properties

   • 0.5 µL can be used if the sample has 10 mm equivalent absorbance values of 3.0 or higher (>150 ng/ µL dsDNA)

   • Do not use samples with dye!

5. Lower sampling arm (has upper pedestal)

6. Click Measure IMMEDIATELY after lowering sampling arm

7. Make sure drop of liquid (sample column) was touching both the lower pedestal and the upper pedestal of the sampling arm

8. When done with measurement wipe both the upper and lower pedestals with a lint-free laboratory wipe

9. Use a fresh pipette tip and a fresh drop of sample everytime. Failure to do so will result in increased concentration measurements due to evaporation.

10. If measuring many samples take a new blank every 30 minutes

11. When done clean with dH₂O and a lint-free laboratory wipe

Troubleshooting unusual spectra:

• Negative absorbance values: the pedestal is dirty or there was something wrong with the blank➝ clean the pedestals and take a new blank measurement

• Ragged appearance (lines not smooth): bad blank➝ clean pedestal and redo blank

• Jagged appearance (very rough lines almost like a zigzag): broken sample column➝ clean and recondition both pedestals and try increasing sample volume (no more than 2 µL)

• High 230 nm absorbance value relative to sample: contaminated sample➝ may need to redo sample

Instructions for interpreting the NanoDrop results for DNA are found here:

https://docs.google.com/document/d/1hsAzy2MWxDj2EdsFWOlJUFh-A3jtAZ4Cf6RnZnpHLRA/edit

Measuring Proteins

1. Clean and recondition pedestal surfaces prior to starting

   • Cleaning

     • Use dH₂O and a lint-free laboratory wipe to clean upper and lower pedestals

     • Do NOT spray or squirt water onto NanoDrop

     • If there is a dried sample stuck on the pedestals use a 3 µL drop of HCl followed by 3 µL of dH₂O to clean

   • Reconditioning

     • Use the applicator in the reconditioning kit, take a pin-head sized amount of NanoDrop Pedestal Reconditioning Compound (PR-1)

     • Apply a thin even layer of PR-1 to the flat part of the lower and upper pedestals

     • Wait 30 seconds for the PR-1 to dry

     • Fold a lint-free laboratory wipe into quarters and wipe PR-1 off until no more dark PR-1 compound residue shows up on the wipe

2. Take a blank measurement using the same buffer as used in the sample

   • Many common buffers are not suitable for measurement

   • To check if the buffer is suitable:

     • Pipette 2 µL of dH₂O onto lower pedestal

     • Lower sampling arm

     • Click Blank

     • Wipe pedestals with a dry, lint-free wipe

     • Pipette 2 µL of blank buffer onto lower pedestal

     • Lower sampling arm

     • Click Measure

     • Wipe pedestals with a lint-free laboratory wipe

     • Analyze spectra- should be a straight line that vaies no more than 0.4 mm from baseline

     • If buffer is compatible continue

     • Pipette 2 µL of a new sample of blank buffer onto lower pedestal

     • Lower sampling arm

     • Click Blank

   • If it is already known that the buffer is suitable:

     • Pipette 2 µL of blank buffer onto lower pedestal

     • Lower sampling arm

     • Click Blank

3. Enter sample name in Sample ID box and choose appropriate sample type

   • Enter extinction coefficient and/or molecular weight if asked (see guide)

4. Lightly vortex sample

   • Note protein sample must be purified prior to measurement

5. Pipette 2 µL of the sample onto the lower pedestal

6. Lower sampling arm

7. Click Measure IMMEDIATELY after lowering sampling arm

8. Make sure drop of liquid (sample column) was touching both the lower pedestal and the upper pedestal of the sampling arm

9. When done with measurement wipe both the upper and lower pedestals with a lint-free laboratory wipe

10. Use a fresh pipette tip and a fresh drop of sample everytime. Failure to do so will result in increased concentration measurements due to evaporation.

11. If measuring many samples take a new blank every 30 minutes

12. When done clean with dH₂O and a lint-free laboratory wipe

Troubleshooting unusual spectra:

• Negative absorbance values: the pedestal is dirty or there was something wrong with the blank➝ clean the pedestals and take a new blank measurement

• Ragged appearance (lines not smooth): bad blank➝ clean pedestal and redo blank

• Jagged appearance (very rough lines almost like a zigzag): broken sample column➝ clean and recondition both pedestals and try increasing sample volume (no more than 2 µL)

• Jagged appearance at the top of the 280 nm peak: sample too concentrated➝ dilute sample and redo all measurements