Classes / Training

[Background music begins]

[Visual: The camera pans over a lab bench laid out with supplies for the Nanodrop assay.]

Narrator:

We first want to get set up with the supplies you need to bring to the core facility. Bring your sample, pipette tips, chem wipes, and a pipeter that you can set to one or two microliters.

[Visual: The Nanodrop One instrument is shown on a lab bench. A gloved hand points to available assay options on the software interface.]

Narrator:

The NanoDrop One can quantify many sample types: double-stranded DNA, single-stranded DNA, RNA, micro-array with two fluorophores, custom factors, oligo-DNA, or oligo RNA.

[Visual: The assay type is selected and the blue lights on the instrument indicate it is completing a self-test. The gloved hand raises the instrument arm, wipes the pedestal with a chem wipe, and then pipets liquid onto the instrument pedestal.]

Narrator:

We first need to create a blank using our sample diluent. Begin by selecting your sample type. The NanoDrop will perform an instrument self-test. To load a blank or sample onto the NanoDrop, begin by raising the NanoDrop arm. Using a dampened Kim wipe, dab the lower and upper pedestal to clean away any prior sample. You'll then apply one or two microliters of either your diluent or your sample to the lower pedestal and lower the upper arm.

[Visual: The pedestal is shown close up while a gloved hand pipets liquid onto it. The instrument arm is lowered to show creation of a liquid column.]

Narrator:

If we look at that more closely, we can see the drop of sample on the hydrophobic lower pedestal and the column created by lowering the arm.

[Visual: The instrument screen is shown and the blank option selected. A new sample is pipetted and the instrument arm lowered to automatically read the absorbance spectra.]

Narrator:

Press blank in the lower left of the screen to begin measuring.

Repeat the process with each of your samples. The NanoDrop will automatically begin measuring when the arm is lowered.

[Visual: The absorbance spectra of a sample is shown. The narrator scrolls right on the software interface to show a table of absorbance values at specific wavelengths.]

Narrator:

The NanoDrop will display your absorbance across its entire spectral range. Of interest are peaks at 230, 260, and 280. While absorbance at 230 indicates organic compounds such as phenol are present, the ratio of 260 to 280 absorbance is indicative of each sample type. Scrolling right allows us to see the measurement for each of these absorbance values.

[Visual: The gloved hand selects end experiment.]

Narrator:

When you have read all of your samples, press end experiment. If a USB is inserted, you'll see it here and can export your data.

After exporting, press end experiment.

[Visual: The gloved hand uses a chem wipe to clean the instrument pedestal. Red text appears reminding users to apply water to the chem wipe and not directly to the instrument.]

Narrator:

Before leaving, clean the pedestals one last time using a chem wipe dampened with distilled water. 

[Background music fades]

[Background music begins]

[Visual: The camera pans over a lab bench laid out with supplies for the Qubit assay.]

Narrator:

Bring to the core facility your sample, pipette tips, and pipeters. We will provide a dilution buffer, standards, fluorometric reagent, Qubit-specific assay tubes, and a quick start guide.

[Visual: Gloved hands pipette reagents into microcentrifuge tubes.]

Narrator:

To prepare your sample, we'll begin by making a master mix of our dilution buffer and fluorometric reagent. For each sample, you'll need 199 microliters of dilution buffer.

Make sure to include enough for excess and the two standards.

To the dilution buffer, add our 200x fluorometric reagent: one microliter for each of your samples, the two standards, and however much excess you've chosen to make.

[Visual: Gloved hands swirl the tube of mastermix and aliquot the mastermix into sample tubes. Assay standards and samples are pipetted into the associated sample tubes.]

Narrator:

Swirl the master mix.

For each of the standards, add 190 microliters of fluorometric master mix to each tube. For your samples, you'll add between 190 and 199 microliters of the master mix, depending upon the dilution that you've chosen to use.

For each standard, add 10 microliters — 10 microliters of standard one into the standard one tube, and 10 microliters of standard two into the standard two tubes. Dilutions may use between 1 and 10 microliters, depending upon how concentrated you anticipate your sample being.

[Visual: Gloved hands use a marker to label assay tubes. Tubes are vortexed. A visual timer is shown indicating the passage of two minutes.]

Narrator:

Make sure you label each tube. This is particularly important if you are measuring multiple samples. Vortex each sample to combine well.

Then incubate for two minutes.

[Visual: The Qubit instrument is shown. A gloved hand selects the DNA assay type.]

Narrator:

When you're ready to quantify your samples on the Qubit fluorometer, begin by selecting your assay type.

Next, you will make a two-point standard curve.

[Visual: Sample tubes are inserted sequentially into the Qubit instrument and the read button selected for each one.]

Narrator:

Insert standard one and press read.

Then insert standard two and press read again.

You're now ready to begin reading each of your samples. Insert your first sample and press read.

[Visual: Software options for calculating stock concentration and viewing data are selected.]

Narrator:

To calculate your stock concentration, select calculate stock concentration, adjust the volume of sample used, and save the calculation. You can continue reading samples by pressing read next sample.

[Visual: The instrument screen shows data plotted on the standard curve. A thumb drive is inserted into the instrument and the export data option selected.]

Narrator:

The fluorescent absorbance of your sample is plotted on the two-point standard curve. The assay tube concentration does not take into account your dilution factor, so be sure to use the calculate function. When you have read your last sample, press data on the lower right-hand side of the screen. You can export to the Invitrogen thumb drive using the thumb drive button.

Press home twice to return to the main screen.

[Background music fades]

[Background music begins]

[Visual: A cursor selects the Genesys software icon. The software menu opens.]

Narrator:

The G-Box is controlled using the Syngene Genesys software on the computer to the left of the digital darkroom.

To access the Genesys software, select the icon to the upper left of the computer screen.

[Visual: A cursor indicates the location of the darkroom status indicator then hovers over assay selection options.]

Narrator:

The status of the digital darkroom can be found to the bottom of the screen. Hovering over the digital darkroom icon allows you to see details of the status. A green check mark indicates that the dark room is ready for use. The G-Box is capable of capturing images from a variety of gel or blot types: DNA agarose gels, DNA acrylamide gels, RNA agarose gels, RNA acrylamide gels, visible protein gels, fluorescent protein gels, and stain-free gels. For blots, there is a chemi rapid and a manual chemi blot option, as well as chemi blot in series with possible signal accumulation calculator, fluorescence blot, visible blots, and stain-free blots. We can delve into any of these menu options to get more details about what are available on our instrument.

[Visual: The agarose gel option is selected and a submenu opens.]

Narrator:

For example, agarose gels allow use of ethidium bromide, gel green, GelStar, propidium iodide, and more. Deselect the recent selections option to the bottom right to see all possible options on our instrument.

[Visual: The fluorescent gel option is selected and a submenu opens.]

Narrator:

This is of particular interest for fluorescence, as our instrument allows use of Alexophlores from 350 at the blue end of the emission spectrum to 750, all the way up at the near infrared emission spectrum.

[Visual: The submenu is exited and the screen returns to the main menu. The cursor selects gel options. The chemi rapid assay type is selected. The image exposure screen opens.]

Narrator:

To capture your data image, select either gel or blot from the menu. Chemi rapid is the most commonly used menu item. The digital dark room will immediately begin an exposure and ask for any needed anti-reflective screens. To stop this process and unlock the digital darkroom, select stop.

[Visual: Live imaging on the software screen shows placement of an antireflective screen and blot into the imaging system.]

Narrator:

You can then insert any needed anti-reflective screens and your gel or blot, using the white light camera to position it accurately.

[Visual: The digital darkroom is shown on the lab bench. The door is opened and a gloved hand inserts an antireflective screen and blot into the imager before closing the darkroom door.]

Narrator:

The button to the right-hand side of the digital dark room is used to open the door. Anti-reflective screens are found on the right-hand side on the countertop. Insert the correct anti-reflective screen and position your gel or blot again using the white light camera, then gently close the digital dark room door. Exposure calculations will immediately begin and can be seen on the status icon.

[Visual: The software screen is again shown. The cursor makes adjustments to the image exposure settings and a final exposure of the gel is shown.]

Narrator:

Select your image to see the final exposure.

Sliding bars to the left-hand side of the screen can be used to zoom in, pan around your image to see things in detail, and then zoom back out. Other sliding bars can be used to adjust the white point or the black point to accurately affect both brightness and contrast.

[Visual: The cursor selects the save option and navigates selection of a destination folder for the image file.]

Narrator:

Be sure to save your images before exiting. Use save to save your data as a SGD file. This is Syngene's proprietary file type that contains all of your metadata. You may save your file either to a folder that has a date and investigator name, or to your own thumb drive. Folders saved to this computer will be deleted each month, so be sure to back them up as soon as possible.

You may also export your images using save as. Images can be exported as either TIFFs, BMPs, or JPEGs, though JPEGs are not recommended as compression results

in data loss. Also be sure to increase your resolution to a minimum of 300 dots per inch to ensure your images have adequate resolution for printing. Again, save either to a folder with your name and date or to your own thumb drive, and give your file a name that you can remember.

[Visual: Camera pans out to show G:Box digital darkroom and imaging computer on a lab bench.]

Narrator:

You are now ready to analyze your data using Genetools software on either the core facility computer or your own PC.

[Background music fades]