TITLE: Quantifying and Checking Genomic DNA
AUTHORS: Carlos Goller, Carly Sjogren

[DESCRIPTION]
Overview and Goals
Your bacterial isolate has been grown on agar plates, lysed open and genomic DNA has been isolated for sequencing. Next, we need to quantify our DNA yield and assess its integrity. For this, we will use the Nanodrop spectrophotometer and Qubit to measure our DNA quantity, We will also use an Agilent TapeStation electrophoresis system to assess the size of the DNA and quality.

After completing this lab you will gain the following lab skills:

Lab safety and proper personal protective equipment (PPE)
Proper use of a Nanodrop spectrophotometer and Qubit fluorometer to analyze DNA samples.
Analysis of DNA concentration and integrity using absorbance, fluorescence, and gel electrophoresis.

[BEFORE_START]
Review the protocols and figures below to learn about how the NanoDrop spectrophotometer, Qubit fluorometer, and TapeStation electrophoresis systems work. 


[STEPS]
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
1. Based on the NanoDrop ND-1000 Spectrophotometer Protocol.
 

Before starting, watch this 10-minute video: NanoDrop Microvolume Quantitation of Nucleic Acids
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
2. Initializing
 
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
2.1. There will be a laptop next to the NanoDrop. Log in using your NCSU ID and password.


SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
2.2. Once you’ve logged in, select the ND-1000 program from the selection of apps on the left side of the screen.
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
2.3. In the main menu, select “Nucleic Acid” from the left column, which will take you to the screen on the right. This is where you’ll see and record results.

SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
2.4. The machine will initialize and may make some noises; do not be alarmed.
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
2.5. Once you have set up the NanoDrop program on the laptop, you’ll be prompted to load a water sample to clean and initialize the NanoDrop. 
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
3. Blanking
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
3.1. Once the machine has been initialized, lift up the bar from the pedestal and ensure the pedestal is clean by wiping each side with a KimWipe. 
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
3.2. Next, you will need to load a “blank” sample of buffer. This is to calibrate the machine to the particular buffer your DNA is housed in. 
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
3.3. Using a p10 add 1 µL -2 µL  of your DNA solvent to the lower pedestal (see image below) and gently lower the NanoDrop arm.

SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
3.4. On the computer screen, select “Blank” at the top of the screen after loading.
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
3.5. Next measure your blank by selecting “Measure” at the top of the screen after loading.

The results of the blank should come back near 0 for each metric. If not, clean off the pedestal thoroughly and then load and measure another blank. Once you’ve successfully measured your blank, you may measure your sample.
 




SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
4. Blanking
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
4.1. Using a 10 µl filtered pipette tip, add 2 µL  of your sample to the center of the pedestal.

 

SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
4.2. Close the bar, and then look to make sure the droplet is touching both the upper and lower measurement pedestals as on the right.
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
4.3. After ensuring the sample is properly placed, press measure in the top left corner of the screen. 

 

SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
4.4. For each read, record the ng/µl and 260/280 values in your spreadsheet, in addition to whatever other information your instructor requests.
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
4.5. Once the measurement has been taken and data recorded, open the bar and wipe off each side of the pedestal with a KimWipe. 
 

SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
4.6. Only after cleaning the pedestal can you start to add another sample.
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
4.7. After finishing your last read, clean off the pedestal, close the NanoDrop program, and log off of the station laptop.
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
5. Recording you DNA quantifications
SECTION: Activity 1:Quantification of DNA with the Nanodrop Spectrophotometer
5.1. Create an Excel or Google spreadsheet to keep track of your data from NanoDrop. Title it with the date and the name of your samples. Record the following values:
the concentration of your DNA in ng/µl, 
the ratio of the absorbance at 260 and 280. DNA should have a ratio of 1.8; if RNA is present, the ratio will be higher. If protein is present, the ratio will be lower than 1.8.
230/260 ratio that is used to learn about other substances that may be in your sample
SECTION: Activity 2: DNA Quantification with Qubit
6.  
Follow the link, scroll to the bottom of the webpage and watch this video about the Qubit fluorometer: Qubit™ 4 Fluorometer video (4:29 min) 


 

SECTION: Activity 2: DNA Quantification with Qubit
6.1. Label the tube lids with a Sharpie. Label 3 tubes (2 for standards and 1 for your sample): 
"1" (standard)
"2" (Standard)
Bacterial species number, Ex: "105" (sample)


 


SECTION: Activity 2: DNA Quantification with Qubit
6.2. Prepare your standards to calibrate your sample measurement: 
Each standard tube requires 190 µL of Qubit™ working solution
Add 10 µL  of each Qubit™ standard to the appropriate tube. 
The final volume in each tube must be 200 µL 

SECTION: Activity 2: DNA Quantification with Qubit
6.3. Prepare your sample:
Each sample tube requires 195 µL  of Qubit™ working solution
Add 5 µL  of each user sample to the appropriate tube using a p10. 
The final volume in each tube must be 200 µL 


SECTION: Activity 2: DNA Quantification with Qubit
6.4. Mix each sample by vortexing for 5 s . 

SECTION: Activity 2: DNA Quantification with Qubit
6.5. Allow all tubes to incubate at Room temperature for 2 min .
 

SECTION: Activity 2: DNA Quantification with Qubit
6.6. Calibration of the Qubit™ Fluorometer requires the standards to be inserted into the instrument in the right order. Read the prompts from the Qubit to insert the standards in the correct order. Note: we will assist you through this process. The information provided below is so you can review the entire process. 
On the Home screen of the Qubit™ Fluorometer, press the 1X dsDNA High Sensitivity (HS) assay icon. The “Read standards” screen is displayed. Press Read Standards & run samples to proceed. Note: If you have already performed a calibration for the selected assay, the instrument prompts you to choose between reading new standards and running samples using the previous calibration. If you want to use the previous calibration, press Run samples and skip to step 2.4. 
Insert the tube containing Standard #1 into the sample chamber, close the lid, then press Run standards. When the reading is complete (~3 seconds), remove Standard #1.
Insert the tube containing Standard #2 into the sample chamber, close the lid, and then press Read standards. When the reading is complete, remove Standard #2. The instrument displays the graphical results on the Standards complete screen. 
Press Next from the Standards complete screen to read your Samples. 
In the Sample volume screen, enter the sample volume added to the assay tube (from 1-20 µL). Enter the volume directly in the sample volume text box, use the + or – buttons or adjust the sample volume wheel to select the sample volume added to the assay tube. Note: The sample volume used (1-20 µL) changes the assay accuracy range. A different sample volume or assay may be required if the sample concentration is outside of what the assay can accurately quantify. 
Insert a sample tube into the sample chamber, close the lid, then press Run sample. When the reading is complete (~3 seconds), remove the sample tube. 
SECTION: Activity 2: DNA Quantification with Qubit
6.7. Record your concentrations in ng/µl in your notes and directly on the tubes of your genomic DNA samples.
SECTION: Activity 3: Genomic DNA Assessment with TapeStation
7.  
The Agilent TapeStation is an automated gel electrophoresis system that allows users to separate nucleic acids to obtain information about their size and distribution. 
SECTION: Activity 3: Genomic DNA Assessment with TapeStation
7.1. Work with your instructors to prepare samples for analysis.
SECTION: Activity 3: Genomic DNA Assessment with TapeStation
7.2. Allow the Genomic DNA Reagents to equilibrate at room temperature for 30 min . 

SECTION: Activity 3: Genomic DNA Assessment with TapeStation
7.3. Flick the Genomic DNA ScreenTape device and insert it into the ScreenTape nest of the TapeStation instrument. 
Select required sample positions in the TapeStation Controller software. The required consumables (tips, further ScreenTape devices) are displayed in the TapeStation Controller software. 
SECTION: Activity 3: Genomic DNA Assessment with TapeStation
7.4. Vortex reagents and samples. Spin down before use. 
SECTION: Activity 3: Genomic DNA Assessment with TapeStation
7.5. Prepare ladder: 
Pipette 10 µL Genomic DNA Sample Buffer and 1 µL   Genomic DNA Ladder at position A1 in a tube strip. 

SECTION: Activity 3: Genomic DNA Assessment with TapeStation
7.6. For each sample, pipette 10 µL  Genomic DNA Sample Buffer and 1 µL  DNA sample in a tube strip 

SECTION: Activity 3: Genomic DNA Assessment with TapeStation
7.7. Apply caps to tube strips .
SECTION: Activity 3: Genomic DNA Assessment with TapeStation
7.8. Mix liquids using the IKA MS3 vortexer at 2000 rpm for 1 min  

SECTION: Activity 3: Genomic DNA Assessment with TapeStation
7.9. Spin down samples and ladder for 1 min 

SECTION: Activity 3: Genomic DNA Assessment with TapeStation
7.10. Sample Analysis 
Load samples into the TapeStation instrument. 
Place ladder in position A1 on the tube strip holder. 
Carefully remove caps of tube strips. 
Visually confirm that liquid is positioned at the bottom. 
Click Start. 
The TapeStation Analysis software opens automatically after the run and displays results.
SECTION: Activity 3: Genomic DNA Assessment with TapeStation
8.