Feb 22, 2024
Treatment of DNA with cisplatin
This protocol is a draft, published without a DOI.
- 1Wellcome Sanger Institute
Protocol Citation: Martin Pollard 2024. Treatment of DNA with cisplatin. protocols.io https://dx.doi.org/
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: July 19, 2023
Last Modified: February 22, 2024
Protocol Integer ID: 85238
Keywords: promega human dna with cisplatin, treating promega human dna, cisplatin, treatment of dna, dna, treatment
Abstract
A protocol for treating Promega Human DNA with Cisplatin.
Guidelines
Dispose of excess cisplatin safely according to guidance received in SDS.
Materials
25mg cis-Diammineplatinum(II) dichloride - Sigma Aldrich Product Number P4394
0.9% NaCl solution
Promega Human DNA - Human Mixture
Buffer
Gibson P20 pipette
Wide bore tips
a 1.8 mL Eppendorf tube .
Safety warnings
Cisplatin safety warnings:
H300 Fatal if swallowed.
H315 Causes skin irritation.
H317 May cause an allergic skin reaction.
H319 Causes serious eye irritation.
H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled.
H335 May cause respiratory irritation.
H350 May cause cancer.
Measure out in fume cupboard or enclosed chemical preparation unit; add solvent in Biosafety cabinet
Preparation of Cisplatin liquid
Safety information
Perform this step in a fume hood or use an enclosed scale to minimise exposure.
It is preferable to purchase cisplatin in pre-weighed out quantity where possible.
Weigh out 25 mg of cisplatin into 1000 mL of 0.9% NaCl and mix thoroughly to make a stock solution.
Store stock at 2-8 °C and protected from light to avoid precipitation.
Dilute 1 mL of your stock cisplatin solution into 99 mL of 0.9% NaCl and mix thoroughly.
Store diluted cisplatin at 2-8 °C and protected from light to avoid precipitation.
Treatment of DNA with Cisplatin
1h
Pipette 8.811 µL of Sample from stock tube at 227 µg/ml to a 1.8 mL Eppendorf tube to achieve 2 µg of DNA.
Safety information
Perform this step in a fume hood
Add 7.27 µL of diluted cisplatin at 0.83 micromolar (µM) to the tube
Incubate for 16:00:00 in the dark at 37 °C .
16h
Perform a SPRI bead clean-up (as previously described) to remove lingering cisplatin from supernatant.
Add 0 µL of 0 Mass Percent SPRI beads to tube and 20% polyethylene glycol (PEG), 2.5 M NaCl buffer.
Attach tube to magnet and remove supernatant.
Resuspend DNA in 40 µL of buffer to achieve concentration of 50 ng/µl
Protocol references
Fisher, S., Barry, A., Abreu, J. et al. A scalable, fully automated process for construction of sequence-ready human exome targeted capture libraries. Genome Biol 12, R1 (2011). https://doi.org/10.1186/gb-2011-12-1-r1
Eastman, A. (1983). "Characterization of the adducts produced in DNA by cis-diamminedichloroplatinum(II) and cis-dichloro(ethylenediamine)platinum(II)." Biochemistry 22(16): 3927-3933.