Nanodrop Protocol

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==Nanodrop ND-1000 Spectrophotometer Directions==
==Nanodrop ND-1000 Spectrophotometer Directions==
-
#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!)
+
#you do not 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!)
-
#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)
+
#pipet '''4uL of autoclaved dH2O''' onto the lower metal post and gently put the upper arm down (do not push down on it and do not grab it by the black fiber-optics cord)
-
#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
+
#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
-
#pipet '''2μL of autoclaved dH2O''' onto the lower metal post and put the upper arm down
+
#pipet '''2uL of autoclaved dH2O''' onto the lower metal post and put the upper arm down
#double click on the ND-1000 icon on the desktop of the computer
#double click on the ND-1000 icon on the desktop of the computer
-
#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)
+
#when the ND-1000 window opens, click on Nucleic Acid to check concentration and purity of DNA/RNA
-
#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
+
##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
 +
#the prompt will ask you to load a sample (this is the 2uL of water you added), then click ''OK'' and the spectrophotometer should make a clicking noise to let you know it is working
#wipe off dH2O from both posts with a clean part of the kimwipe and '''load your blank'''
#wipe off dH2O from both posts with a clean part of the kimwipe and '''load your blank'''
##use as many uL of this as you expect to use of your sample to measure
##use as many uL of this as you expect to use of your sample to measure
##this is whatever your sample is in; usually dH2O again, but sometimes TE, ethanol, or another buffer
##this is whatever your sample is in; usually dH2O again, but sometimes TE, ethanol, or another buffer
-
#'''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
+
#'''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
#Gently flick your samples to mix and briefly spin down (''homogeneous sample is required for accurate readings!'')
#Gently flick your samples to mix and briefly spin down (''homogeneous sample is required for accurate readings!'')
#To take a sample measurement:
#To take a sample measurement:
-
##'''load 1-2μL of your homogeneous sample''' onto the lower metal post and put the upper arm down
+
##'''load 1-2uL of your homogeneous sample''' onto the lower metal post and put the upper arm down
-
##'''type in sample ID''' into white �Sample� box on screen
+
##'''type in sample ID''' into white ''Sample'' box on screen
-
##'''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)
+
##'''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)
-
##ng/μL in green box at the bottom of the window (manual shows what curves should look like�take general notes on curves)
+
##ng/uL in green box at the bottom of the window
-
##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)
+
###manual shows what curves should look like
 +
###take general notes on curves
 +
##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 2uL of the appropriate blank liquid (same as steps 8-9)
##'''wipe down both posts with a new part of the kimwipe''' and repeat steps 1-6 under "to take a sample measurement" section for all samples
##'''wipe down both posts with a new part of the kimwipe''' and repeat steps 1-6 under "to take a sample measurement" section for all samples
-
#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
+
#pipet '''4uL 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
-
#hit the â��Show Reportâ�� button and type in a name for the sheet, usually in the format:  initials, date, informational line (eg. SEJ_12June07_DNAbox3)
+
#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)
-
#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.
+
#hit print report (this goes to the printer in Marks office and can be pasted in the FarmerLab Molecular work lab notebook if it is after a DNA extraction, or in the persons notebook who is measuring their PCR products, gel extractions, etc.
-
#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)
+
#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)
#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)
#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)
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==DNA Notes==
==DNA Notes==
-
*260/280 should be ~1.80 (otherwise, it�s contaminated: phenol, protein, other)
+
*260/280 should be ~1.80 (otherwise, it is contaminated: phenol, protein, other)
*260/230 should be ~1.8-2.2 (otherwise, co-purified contaminants)
*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)
*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)
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*Generally use the RNase-free water next to the Nanodrop as a blank
*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)
+
*260/280 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
*260/230 should be similar to the range stated for DNA, but this is not usually as important as the 260/280 ratio

Current revision as of 20:42, 6 March 2009

Contents

Nanodrop ND-1000 Spectrophotometer Directions

  1. you do not 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 4uL of autoclaved dH2O onto the lower metal post and gently put the upper arm down (do not push down on it and do not 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 2uL 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
    1. the default is set for double stranded DNA; says DNA-50 by sample type green window under Exit button
    2. 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 2uL 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. Gently flick your samples to mix and briefly spin down (homogeneous sample is required for accurate readings!)
  11. To take a sample measurement:
    1. load 1-2uL of your homogeneous 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/uL in green box at the bottom of the window
      1. manual shows what curves should look like
      2. 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 2uL 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 1-6 under "to take a sample measurement" section for all samples
  12. pipet 4uL 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
  13. 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)
  14. hit print report (this goes to the printer in Marks office and can be pasted in the FarmerLab Molecular work lab notebook if it is after a DNA extraction, or in the persons notebook who is measuring their PCR products, gel extractions, etc.
  15. 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)
  16. 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 is 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


Related Farmer Lab Wiki Links


Nanodrop Manufacturer Info

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