Luckau Protocols:NanoDrop: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(New page: ===NanoDrop Protocol=== ====Purpose==== The NanoDrop is used to quantify genetic material. Although there are many different methods to quantifying DNA, the NanoDrop is a spectrophotometer...) |
No edit summary |
||
| (14 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
===NanoDrop Protocol=== | {|{{table}} width="900" | ||
|- | |||
| width="600" style="background-color: #BCED91;" align="center"| <span style="font-size:32px;"> NanoDrop Protocol </span> | |||
|align="left" style="background-color: #9DB68C;" | | |||
<span style="font-size:16px;"> [[User:Tara_K._Luckau | Tara K. Luckau's Home Page]] </span> | |||
[[User:Tara_K._Luckau/Notebook/Team_ConGen | Conservation Genetics Lab Notebook]] | |||
[[Luckau_Protocols | Tara's Protocols]] | |||
|} | |||
==Purpose== | |||
The NanoDrop is used to quantify genetic material. Although there are many different methods to quantifying DNA, the NanoDrop is a spectrophotometer that calculates absorbance of a sample across different wavelengths. | The NanoDrop is used to quantify genetic material. Although there are many different methods to quantifying DNA, the NanoDrop is a spectrophotometer that calculates absorbance of a sample across different wavelengths. | ||
==Protocol== | |||
# Assemble cafeteria tray | # Assemble cafeteria tray | ||
#* Kim Wipes | #* Kim Wipes | ||
| Line 15: | Line 29: | ||
#*[[Image:NanoDrop1000.jpg]] | #*[[Image:NanoDrop1000.jpg]] | ||
# Open the NanoDrop software, "ND1000" on the desktop | # Open the NanoDrop software, "ND1000" on the desktop | ||
# Choose "Nucleic Acids" | #* Choose "Nucleic Acids" | ||
# Initialize the instrument | # Initialize the instrument | ||
#* Place 2µL of NanoPure water on the pedestal | #* ensure upper and lower pedestal surfaces are clean by wiping with Kim Wipe | ||
#* | #* Place 2µL of NanoPure water on the lower pedestal | ||
#* when it's done, wipe | #* Lower the sampling arm and press OK | ||
#* when it's done, wipe upper and lower pedestals with Kim Wipe | |||
# Calibrate the instrument | # Calibrate the instrument | ||
#* Place 2µL of elution buffer on the pedestal | #* Place 2µL of elution buffer on the pedestal | ||
#* For Clark's lab, elution buffer is 10mM Tris-Cl, pH 8.4 | #* For Clark's lab, elution buffer is 10mM Tris-Cl, pH 8.4 | ||
#* Click "Blank" | #* Click "Blank" | ||
#* when it's done, wipe | #* when it's done, wipe upper and lower pedestals with Kim Wipe | ||
# Measure sample | # Measure sample | ||
#* Place 2µL of sample on the pedestal | #* Place 2µL of sample on the pedestal | ||
#* Enter sample ID | #* Enter sample ID | ||
#* Click "Measure" | #* Click "Measure" | ||
#* wipe | #* wipe upper and lower pedestals with Kim Wipe after each sample | ||
#* Re-calibrate the instrument each 10 samples, or so, by going back to step 6 and clicking "Re-Blank" instead of "Blank" | #* Re-calibrate the instrument each 10 samples, or so, by going back to step 6 and clicking "Re-Blank" instead of "Blank" | ||
# Save data | # Save data | ||
#* Click "Show Report" | #* Click "Show Report" | ||
#* Click " | #* Click "Reports", "Save Report" | ||
#* Click "Table | #* Click "Export Report Table Only" | ||
#* save to your USB drive, as "YYYYMMDD_NanoDrop", use "A" "B" "C", etc to designate multiple saved files in the same day | #* save to your USB drive, as "YYYYMMDD_NanoDrop", use "A" "B" "C", etc to designate multiple saved files in the same day | ||
# Re-initialize the instrument each 30-50 samples, by going back to step 3 | # Re-initialize the instrument each 30-50 samples, by going back to step 3 | ||
| Line 40: | Line 55: | ||
# Clean up thoroughly! Other people use this instrument! | # Clean up thoroughly! Other people use this instrument! | ||
# don't forget to enter data into spreadsheet | # don't forget to enter data into spreadsheet | ||
==Purity Assessment== | |||
* ratio of sample absorbance at 230, 260 and 280nm is used to assess sample purity. The ratios and information relevant to our work with DNA is given below: | |||
===260/280 Ratio=== | |||
* used to assess purity of DNA and RNA | |||
* pure DNA: ~1.8 | |||
* <1.8 → residual reagent form extraction, low nucleic acid concentration (<10 ng/µL) | |||
* >1.8 → not an issue! | |||
===260/230 Ratio=== | |||
* used as a secondary measure of nucleic acid purity | |||
* pure nucleic acid: 2.0-2.2 | |||
* <2.0 → residual guanidine | |||
* >2.2 → blanked on dirty pedestal, inappropriate blank (should be same pH and ionic strength) | |||
==Resources== | |||
* NanoDrop User's Manual [[Media:NanoDrop1000-v3.7-UsersManual.pdf]] | |||
* [http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf NanoDrop Technical Bulletin T042: Assessment of Nucleic Acid Purity] | |||
Latest revision as of 15:28, 10 June 2013
| NanoDrop Protocol |
Purpose
The NanoDrop is used to quantify genetic material. Although there are many different methods to quantifying DNA, the NanoDrop is a spectrophotometer that calculates absorbance of a sample across different wavelengths.
Protocol
- Assemble cafeteria tray
- Kim Wipes
- 2µL pipette
- pipette tips
- gloves
- USB drive
- water and buffer tubes
- samples
- The NanoDrop is located in North Life Sciences, room 325A
- Open the NanoDrop software, "ND1000" on the desktop
- Choose "Nucleic Acids"
- Initialize the instrument
- ensure upper and lower pedestal surfaces are clean by wiping with Kim Wipe
- Place 2µL of NanoPure water on the lower pedestal
- Lower the sampling arm and press OK
- when it's done, wipe upper and lower pedestals with Kim Wipe
- Calibrate the instrument
- Place 2µL of elution buffer on the pedestal
- For Clark's lab, elution buffer is 10mM Tris-Cl, pH 8.4
- Click "Blank"
- when it's done, wipe upper and lower pedestals with Kim Wipe
- Measure sample
- Place 2µL of sample on the pedestal
- Enter sample ID
- Click "Measure"
- wipe upper and lower pedestals with Kim Wipe after each sample
- Re-calibrate the instrument each 10 samples, or so, by going back to step 6 and clicking "Re-Blank" instead of "Blank"
- Save data
- Click "Show Report"
- Click "Reports", "Save Report"
- Click "Export Report Table Only"
- save to your USB drive, as "YYYYMMDD_NanoDrop", use "A" "B" "C", etc to designate multiple saved files in the same day
- Re-initialize the instrument each 30-50 samples, by going back to step 3
- remember to save your data before your re-initialize
- Clean up thoroughly! Other people use this instrument!
- don't forget to enter data into spreadsheet
Purity Assessment
- ratio of sample absorbance at 230, 260 and 280nm is used to assess sample purity. The ratios and information relevant to our work with DNA is given below:
260/280 Ratio
- used to assess purity of DNA and RNA
- pure DNA: ~1.8
- <1.8 → residual reagent form extraction, low nucleic acid concentration (<10 ng/µL)
- >1.8 → not an issue!
260/230 Ratio
- used as a secondary measure of nucleic acid purity
- pure nucleic acid: 2.0-2.2
- <2.0 → residual guanidine
- >2.2 → blanked on dirty pedestal, inappropriate blank (should be same pH and ionic strength)
