Feb 22, 2024

Public workspaceTreatment of DNA with cisplatin

This protocol is a draft, published without a DOI.
  • 1Wellcome Sanger Institute
Martin O Pollard
Open access
Protocol Citation: Martin O Pollard 2024. Treatment of DNA with cisplatin. protocols.io https://protocols.io/view/treatment-of-dna-with-cisplatin-cxgwxjxe
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
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
Attention
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
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 Amount25 mg of cisplatin into Amount1000 mL of 0.9% NaCl and mix thoroughly to make a stock solution.
Store stock at Temperature2-8 °C and protected from light to avoid precipitation.

Dilute Amount1 mL of your stock cisplatin solution into Amount99 mL of 0.9% NaCl and mix thoroughly.
Store diluted cisplatin at Temperature2-8 °C and protected from light to avoid precipitation.

Treatment of DNA with Cisplatin
Treatment of DNA with Cisplatin
1h
1h
Pipette Amount8.811 µL of SampleSample from stock tube at Concentration227 µg/ml to a 1.8 mL Eppendorf tube to achieve Amount2 µg of DNA.


Safety information
Perform this step in a fume hood
Add Amount7.27 µL of diluted cisplatin at Concentration0.83 micromolar (µM) to the tube
Incubate for Duration16:00:00 in the dark at Temperature37 °C .

16h
Perform a SPRI bead clean-up (as previously described) to remove lingering cisplatin from supernatant.
Add Amount0 µL of Concentration0 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 Amount40 µL of buffer to achieve concentration of Concentration50 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.