TITLE: Quantifying Checking Genomic DNA
AUTHORS: Carlos Goller

[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 IMPLEN nano 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 the different bands.

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

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

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


[GUIDELINES]
Follow the steps carefully and pay attention to your instructor's guidance. Ask questions when necessary. 

[STEPS]
SECTION: Activity 1: Quantification of DNA with the IMPLEN Nano Spectrophotometer
1. Based on the Implen nanospectrophotometer protocol.

 


Before starting, watch this 3-minute video: The NanoPhotometer Introduction Video (3:43 min video).  
SECTION: Activity 1: Quantification of DNA with the IMPLEN Nano Spectrophotometer
2. Turn on the Implen and select "Nucleic Acids."  
Ensure that the arm is down when you turn on the Implen to ensure that it can properly perform its initialization step. Additionally, check to ensure the device is set to measure "dsDNA."


 

 

SECTION: Activity 1: Quantification of DNA with the IMPLEN Nano Spectrophotometer
2.1. The machine will initialize and may make some noises; do not be alarmed.

Note: If you are the first to use the Implen, perform steps 1 and 2. If someone else has already used the Implen, skip to  . 

SECTION: Activity 1: Quantification of DNA with the IMPLEN Nano Spectrophotometer
2.2. Pipet 2 µL of “blank” sample buffer onto the sample window. Lower the arm and click “Blank.” 

SECTION: Activity 1: Quantification of DNA with the IMPLEN Nano Spectrophotometer
2.3. Wipe off the sample window and top sensor with a Kimwipe. 

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 IMPLEN Nano Spectrophotometer
3. Pipet 2 µL of your  onto the sample window. Lower the arm and click “Sample.”

SECTION: Activity 1: Quantification of DNA with the IMPLEN Nano Spectrophotometer
3.1. Record the concentration (given in ng/μL) and the A260/A280 ratio.
SECTION: Activity 1: Quantification of DNA with the IMPLEN Nano Spectrophotometer
4. Wipe off your sample from the sample window and top sensor with a Kimwipe. You may use distilled water sprayed on a Kimwipe and dried with another one to clean the pedestal and mirror.

This is crucial to ensuring accurate and consistent results. Two or three wipes on each side is fine, but be sure it is dry each time before moving onto the next sample. Only after cleaning the pedestal can you start to add another sample.
SECTION: Activity 1: Quantification of DNA with the IMPLEN Nano Spectrophotometer
4.1. Clean the sample window and top sensor by pipetting 2 μL of water onto the sample window, lowering the arm, and gently tapping the arm to make sure that the water contacts the top sensor. Wipe off the pedestal and top sensor with a Kimwipe.
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 198 µL  of Qubit™ working solution
Add 2 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, select either the 1X dsDNA High Sensitivity (HS) or 1X ds DNA Broad Range 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. 
 

SECTION: Activity 1: Quantification of DNA with the IMPLEN Nano Spectrophotometer
4.2. Lower the arm to prevent dust from accumulating on the sample window.
SECTION: Activity 1: Quantification of DNA with the IMPLEN Nano Spectrophotometer
5. Create an Excel or Google spreadsheet to keep track of your data from Implen. 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.