Apr 23, 2022
Preparation of 100mg/ml kanamycin solution
- 1University of Cambridge, Beneficial Bio;
- 2Mboalab, Beneficial Bio
Protocol Citation: Jennifer Molloy, Nadine Mowoh 2022. Preparation of 100mg/ml kanamycin solution. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq7o3kvk5/v1
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: Working
We use this protocol and it’s working
Created: April 22, 2022
Last Modified: April 23, 2022
Protocol Integer ID: 61218
Keywords: Kanamycin, Stocks, Cell Culture
Abstract
Kanamycin is an aminoglycoside antibiotic which binds the 30S ribosomal subunit; causes mis-translation. It is used for the selection of cells containing Kanamycin-resistant plasmids on plates and on liquid media at a working concentration of 50-100 µg/mL.
It is most often prepared as a 1000x working stock of 50 -100 mg/mL that is stored at -20 ºC.
The kanamycin solution is measured and then mixed into a container of liquid or plate media before it has solidified. Cells are then streaked onto the plates or grown in the liquid media. Only cells containing the plasmids resistant to kanamycin will grow.
Materials
Materials
- Disinfection solutions (freshly prepared 10% bleach and 70% alcohol)
- Kanamycin sulphate powder (CAS#: 70560-51-9; Linear Formula: C18H36-37N4-5O10-11 · H2SO4)
- Distilled or deionised water
Equipment
- Electronic balance
- Freezer
Consumables
- Weighing boat
- Spatula
- 10ml Measuring cylinder
- 0.22µm syringe filter
- Sterile 20 ml syringe
- Sterile 50 ml centrifuge tube
- Sterile 1.5 ml microcentrifuge tubes
- p1000 Micropipette
- Sterile 1000 µl Pipette tips
- Waste containers
Safety warnings
Kanamycin sulphate is a hazardous substance (SDS) and may damage fertility or the unborn child. It is highly recommended to weigh out all antibiotics in a fume hood or powder weighing station.
Preparing 1000x kanamycin sulphate stock solution
Preparing 1000x kanamycin sulphate stock solution
20m
20m
Clean work surfaces and work space using freshly prepared 10 % (v/v) bleach, followed by 70 % (v/v) alcohol.
- Using the electronic balance, carefully weigh out 0.4 g of kanamycin sulphate powder (CAS#: 70560-51-9; Linear Formula: C18H36-37N4-5O10-11 · H2SO4) into an appropriate size weighing boat using a spatula.
Safety information
Kanamycin sulphate is a hazardous substance (SDS) and may damage fertility or the unborn child. It is highly recommended to weigh out all antibiotics in a fume hood or powder weighing station.
Carefully tip the powder into a sterile 50 mL centrifuge tube.
Using a 10 mL measuring cylinder, measure out 4 mL of sterile distilled or deionised water.
Pour 2 mL water into the 50 mL centrifuge tube containing the kanamycin powder.
Use the remaining 2 mL to rinse the weighing boat and pouring that into the 15 mL falcon tube to make 4 mL total and mix gently by inversion.
Using the sterile 20 mL syringe, aspirate the solution from the falcon tube
Affix a sterile 0.22 µm micro filter to the end of the syringe and dispense thee solution into a new sterile 15 mL centrifuge tube.
Pipette 1000 µL of the filtered 100 mg/mL Kanamycin solution into 4 x sterile 1.5 mL microcentrifuge tubes.
Close the tubes, label and store them at-20 °C if they are not to be used immediately. This is a 1000x stock of 100 µg/µL Kanamycin which is the normal working concentration in liquid or solid media. The stock can be diluted before use to reach the desired concentration.
Note
Kanamycin sulphate does degrade with time and there can be differences in potency and stability between batches. It is recommended when starting with a new batch to titrate the concentration to ensure 100% of non-resistant bacteria are killed. If the antibiotic properties start to drop off, ideally make a new batch from scratch or titrate to an increased effective concentration.