Nanodrop Protocol

From Protists

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Revision as of 20:09, 6 March 2009

Nanodrop ND-1000 Spectrophotometer Directions

1. you don’t have to wait for the machine to warm up b/c it is always plugged in and attached by USB to the computer, so it is ready to use (DO NOT touch mouse or keyboard with gloves!)
2. pipet 4μL of autoclaved dH2O onto the lower metal post and gently put the upper arm down (don’t push down on it and don’t grab it by the black fiber-optics cord)
3. raise the arm again and wipe off the metal posts with a clean kimwipe…use a different part of the kimwipe each time…do this after every blank/sample
4. pipet 2μL of autoclaved dH2O onto the lower metal post and put the upper arm down
5. double click on the ND-1000 icon on the desktop of the computer
6. when the ND-1000 window opens, click on “Nucleic Acid” to check concentration and purity of DNA/RNA…the default is set for double stranded DNA; says “DNA-50” by “sample type” green window under “Exit” button (do not change this unless you are measuring something else such as single stranded DNA or RNA)
7. the prompt will ask you to load a sample (this is the 2μL of water you added), then click “OK” and the spectrophotometer should make a clicking noise to let you know it is working
8. wipe off dH2O from both posts with a clean part of the kimwipe and load your blank
   1. use as many uL of this as you expect to use of your sample to measure
   2. this is whatever your sample is in; usually dH2O again, but sometimes TE, ethanol, or another buffer
9. hit the “BLANK” button on the top task bar of the ND-1000 window, then wipe off blank with a clean part of the kimwipe and begin taking samples
10. To take a sample measurement:
    1. load 1-2μL of your vortexed sample onto the lower metal post and put the upper arm down
    2. type in sample ID into white “Sample” box on screen
    3. hit “Measure” button and then wait until the clicking is finished and the dialog box in the upper taskbar says “Measurement Complete” (can also check that measurement was taken by clicking on “Show Report” button)
    4. ng/μL in green box at the bottom of the window (manual shows what curves should look like…take general notes on curves)
    5. be sure to check the dialog box in the upper taskbar every 10 or so samples for the message “Make new BLANK Measurement” and re-blank with 2μL of the appropriate blank liquid (same as steps 8-9)
    6. wipe down both posts with a new part of the kimwipe and repeat steps blah through blah under "to take a sample measurement" section for all samples
11. pipet 4μL of autoclaved dH2O onto the lower metal post and gently put the upper arm down…raise the arm again and wipe off the metal posts with a clean kimwipe…use a different part of the kimwipe each time
12. hit the “Show Report” button and type in a name for the sheet, usually in the format: initials, date, informational line (eg. SEJ_12June07_DNAbox3)
13. hit print report (this goes to the printer in Mark’s office and can be pasted in the FarmerLab Molecular work lab notebook if it is after a DNA extraction, or in the person’s notebook who is measuring their PCR products, gel extractions, etc.
14. under the “Report” tab at the very top of the window go under “save report” and hit the 2nd option down from the top (it will save under the Nanodrop file folder under program files in C://, but there is a shortcut to this on the desktop)
15. save all FarmerLab Nanodrop files in the Nanodrop file on the desktop of the FarmerLab Euglena PC in front of the windows (right click on .txt file and open with excel and re-save as an excel file in the same folder and then copy and paste required data into the DNA.xls sheet to go with the respective cell concentration and DNA extraction data for the tube measured)

DNA Notes

• 260/280 should be ~1.80 (otherwise, it’s contaminated: phenol, protein, other)
• 260/230 should be ~1.8-2.2 (otherwise, co-purified contaminants)
• Can do DNA precipitation to clean the DNA (Cleaning DNA & RNA) or if not too far from correct values, should be fine (can also check to be sure that there are still long strands of DNA by running the DNA sample on a gel)

RNA Notes

• Generally use the RNase-free water next to the Nanodrop as a blank
• 260/280 should be >1.80 (~2.0 is considered “pure” RNA)
• 260/230 should be similar to the range stated for DNA, but this is not usually as important as the 260/280 ratio