Feb 22, 2024
Version 2
UV Exposure Protocol V.2
This protocol is a draft, published without a DOI.
- 1Wellcome Sanger Institute
Protocol Citation: Martin O Pollard 2024. UV Exposure Protocol. protocols.io https://protocols.io/view/uv-exposure-protocol-c9kmz4u6Version created by Martin O Pollard
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: February 22, 2024
Last Modified: February 22, 2024
Protocol Integer ID: 95597
Abstract
A procedure utilising a UV crosslinker to expose isolated DNA to UV radiation with the aim of inducing lesions in the DNA typical of that kind of damage induced by UV.
Materials
Human Genomic DNA - Human Mixed - G3041 1 × 100μg
UVP Crosslinker CX-2000
Dilution buffer
1* Gibson P20 pipette
2 * Wide bore tips
1 * Sterile petri dish
1 * Eppendorf tube
Safety warnings
Ultraviolet Crosslinkers are a powerful source of ultraviolet radiation. Even though they are not easily accessible, do not attempt to disengage or override the internal safety interlocks. Exposure to the UV radiation may result. If the UV sources remain on when the door is open, the unit is malfunctioning and use should be discontinued until the unit is serviced. Do not expose unprotected eyes or skin to UV radiation. Always disconnect the UV Crosslinker from its electrical supply before servicing.
UV treatment
UV treatment
Pipette 2.2 µL of Sample which has a measured concentration of 227 Mass Percent from stock tube to sterile plastic petri dish.
Dilute material in petri dish with 22.8 µL of buffer to achieve a target of 25 µL of DNA at 20 Mass Percent yielding 0.5 µg of DNA. Gently mix but do not agitate vigorously.
Expose tube to UV light using Crosslinker at 254 nm to exposing the DNA to 800 J/m2 (80000 μJ/cm2)
Equipment
UVP Crosslinker CX-2000
NAME
UV Crosslinker
TYPE
UVP
BRAND
95-0339-02
SKU
LINK
254nm wavelength 8W
SPECIFICATIONS
Place sample in Crosslinker drawer
Press the ENERGY button, enter the exposure energy of 80000 microjoules/cm2 as 80 on the keypad.
Note: YOUR ENERGY EXPOSURE SETTINGS DISPLAYED MUST BE MULTIPLIED BY
100 to obtain the exposure in microjoules/cm2. If settings are correct, push ENTER on the touch pad.
Press START button to activate crosslinker and wait for cycle to finish.
Transfer sample to eppendorf tube with pipette.
Protocol references
Douki, T. and J. Cadet (2001). "Individual Determination of the Yield of the Main UV-Induced Dimeric Pyrimidine Photoproducts in DNA Suggests a High Mutagenicity of CC Photolesions." Biochemistry 40(8): 2495-2501.
Muylaert, Isabella "Saturation energy of UVC on genomic DNA" personal communication