Hello. I am using a CL-1000 UV crosslinker from UVP to irradiate my reagents for Multiple Displacement Amplification before using them for Whole Genome Amplification.
According to a reference, it is feasible to UV-treat a tube of mastermix on ice with UV Crosslinker at 254 nm for 0, 30, 60 and 90 min. These represent the UV doses of 0, 5.7, 11.4 and 17.1 J/cm2, respectively.
However, the only adjustable parameters on the instrument are Energy and Time. I could not relate the UV doses to the parameters mentioned.
Hence may I know how can i adjust the desired Energy level on the UV crosslinker to achieve the UV doses?
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026161
On a CL-1000 the energy setting default is 1200, which is 120,000 uJ/cm^2, or 0.12J/cm^2. The max energy setting is 9999, which is 999,900uJ/cm^2 or 0.9999J/cm^2. The energy is measured so the same amount is applied each time, but the time my vary (depending on if the tubes are warm, etc.)
This isn't enough for you, so you'll have to use the time setting. However, the time setting can lead to variations in exposure due to tube age/temperature etc.
On my relatively new CL-1000, it took 12 seconds to apply 0.1J/cm^2 (i.e with the energy setting on 1000). Dubiously extrapolating from this, 30 mins of exposure would equate to 15J/cm^2 (not 5.7J/cm^2). Alternatively, 5.7J/cm^2 would have taken 11m 24s.
I don't know how the output of the tubes would vary over 30+ minutes, but I would worry that the J/cm^2 would vary greatly between machines. Even if they measured the value by some more accurate method on their machine, your machine may behave quite differently over that period of time.
What is the dimension of your tube? You may need to consider the optical absorbance of your fluid and tube. The values above would apply only to the surface of whatever is being irradiated, and you may want to ensure that all the fluid receives the same UV dose. Consider the absorbance of the tube material (preferably quartz) and of the fluid. If the fluid is stirred during irradiation you can use an average intensity calculated from optical properties, and multiply by exposure time. If it's not stirred, you will have a distribution of UV intensity and therefore a distribution of UV dose in the fluid. That said, if the optical absorption of the tube wall and the fluid at 254nm are small for their thickness, this won't be an important factor.
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Genetic Analysis