Preparation of 100mg/ml kanamycin solution
Jennifer Molloy, Nadine Mowoh
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.
Steps
Preparing 1000x kanamycin sulphate stock solution
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 0.4g of kanamycin sulphate powder (CAS#: 70560-51-9; Linear Formula: C18H36-37N4-5O10-11 · H2SO4) into an appropriate size weighing boat using a spatula.
Carefully tip the powder into a sterile 50mL centrifuge tube.
Using a 10mL measuring cylinder, measure out 4mL of sterile distilled or deionised water.
Pour 2mL water into the 50mL centrifuge tube containing the kanamycin powder.
Use the remaining 2mL to rinse the weighing boat and pouring that into the 15mL falcon tube to make 4mL total and mix gently by inversion.
Using the sterile 20mLsyringe, 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 15mL centrifuge tube.
Pipette 1000µL of the filtered 100mg/mL Kanamycin solution into 4 x sterile 1.5mL 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 100µg/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.
