We have recently added GreenView Plus fluorescent DNA stain to our online store. Much like GreenView, GreenView Plus DNA gel stain (Applied BioProbes) is a safe, easy-to-use alternative to ethidium bromide and can be used with UV or blue light transilluminators to detect nucleic acids in agarose gels. GreenView Plus also has increased DNA sensitivity and as a result has better detection than GreenView in samples with low DNA concentration.
We have used GreenView Plus in combination with both of our blue (470nm) LED and UV (310 nm) transilluminators, and have also compared detection to staining with GreenView. The main purpose of this blog post is to share some of these images. We have also included some notes on our current imaging set-up.
Electrophoresis and staining procedures
The majority of steps used in the gels shown below are the same. Differences (stain used, amount of DNA and the transilluminator) are mentioned in the gel description.
Gels are 0.8% agarose in TBE buffer
DNA sample used is a 1 Kb DNA ladder (NEB Part #N3232S), 500 ng/µL. An aliquot is further diluted to 50 or 100 ng/µL.
Volume loaded is 5 µL of sample per lane.
Electrophoresis at 100 V for approx. 40 mins with our mini-gel electrophoresis system and power supply
After electrophoresis, gels were transferred to a plastic container with 25mL of gel stain. Gel stains were diluted from 10,000X stock to 2X in 25 mL of TBE buffer.
Gel staining were placed on a platform shaker with gentle shaking for 30 mins.
After staining, transfer gels to a container with water. If necessary, you can destain GreenView Plus gels in water to reduce any background stain (approx 10 mins). GreenView gels do not require any destaining step and can be imaged right away.
Imaging gels with a DSLR camera
To image the gels, we use a Canon T3 DSLR camera. This is mounted onto a camera stand (copy stand) as shown in the images. The transilluminator (blue or UV) is placed in a light-tight box directly below the camera. We are using the desktop imaging enclosure, although any light-tight box or dark-room will work .In the images below, the imaging enclosure is shown open to show the gel and transilluminator.
For capturing the images, we have recently started using DarkTable. Darktable is an open-source photography application that can be used - with compatible cameras - to take pictures directly from your computer, control camera settings (aperture, shutterspeed etc) and manage your images. We also use the live view to position and focus the gel above the camera. Below is a screenshot of the application.