Selection of Hygromycin B-resistant Transformants

Tim Burland (Last Update May 4, 1993)

1. Grow amoebae

After transformation with DNA carrying the hygromycin B phosphotransferase gene, grow amoebae on shaker at 26^o in SDM, maintaining a cell density 2-80x10⁵/ml.  Mean generation time should be 30 hours or less.

2. Make Hygromycin B stock

Warning:  Toxic.

These methods were established using Hygromycin B solid (CalBiochem Cat#400050) at about 1000 units/mg. This product is no longer available. Instead, Hygromycin B is supplied as a solution (CalBiochem Cat#400051) at around 400,000 units/ml. Preliminary expts indicate that the concentration suggested here for selection, 100mg/ml, can be substituted by 100units/ml of the new product.

Filter-sterilize the supplied solution and store at 4^o.

3. Make selective plates (100ug/ml Hyg B)

Make  molten DSDM agar

Accurately measure the quantity of DSDM agar needed

To each litre of accurately measured DSDM molten agar, add 5ml 20mg/ml stock Hygromycin B and mix

Pour plates, let solidify overnight, then bag plates and store in 20^o incubator.

4. Plate amoebae

Count axenically-growing amoebae using haemacytometer

Harvest amoebae by centrifugation at 200 x g for 2 min.

Resuspend in formalin-killed bacteria  (FKB) at 1-50x10⁶ amoebae/200uL.

For axenically-grown amoebae, be sure the FKB are suspended in BSS, not water.

For Viable Counts (if needed):

• Dilute amoebae x104 through SDM
• Spread about 100 cells (judged by haemacytometer count) with 150ml FKB in duplicate on DSDM

For selection for Hygromycin B resistance:

• Spread 200uL undiliuted cells in FKB (0.5-50x10⁶ amoebae) onto each 100ug/ml HygB-DSDM plate
• Incubate all plates 26^o
• Score viable counts after 5 & 7 days
• Score HygB selections after 10-21 days.