Race tube assay

Abstract

The aim of this protocol has been to investigate the recovery abilities of wt Neurospora strains and strains lacking the main clock components from starvation by quantifying their growth rate.
 

Background

Race tubes are widely used for quantification of growth rate of Neurospora strains across the research field of circadian rhythms. The current protocol has been established to present a simple method for quantifying the recovery abilities of wt and clock-mutant strains under various conditions. 
 

Materials and Reagents

1. Biological Materials

   1.1. Wt (Fungal Genetics Stock Center (FGSC) #1858), ∆wc1 (1.) and frq¹⁰ (FGSC #7490) strains of Neurospora crassa

    

2. Reagents

   2.1. Vogel’s medium (2.)

   2.2. L-arginine (AppliChem #A1700.1000)

   2.3. D-glucose (BioLab #DEX11000)

   2.4. Biotin (Sigma Aldrich #B4501)

   2.5. Agar (BioLab # BAA10500)

Equipment

1. Infors HT Minitron incubator
2. Race tubes (glass growth tubes of approximately 40cm length and 16mm diameter bent at both ends to hold agar media and limit the growth of the strain to one direction)
3. Ruler (to measure the growth distance)

Procedure

1. Race tubes containing 12ml of race tube medium (Vogel’s medium supplemented with 0.17% L-arginine, 3.2% agar, 2% glucose and 10 ng/ml biotin) were pre-incubated at 25°C.  Starved and normal mycelia were initially grown in liquid cultures at 25 °C.
2. Race tubes were inoculated with mycelia originated from starved or normal cultures at one end of the pre-incubated tube, so that the culture could grow across the agar surface at 25 °C. 
3. Growth rate on race tubes was determined in three intervals (0–6 hr, 6–12 hr, 12–24 hr) during the first day following inoculation.

Data analysis

Growth rate has been calculated based on the measured growth distance during the specific time intervals (0–6 hr, 6–12 hr, 12–24 hr). Growth rate of the samples originated from starved cultures was normalized to those of the non-starved samples of the same strain during the same period. Analysis of the data can be found in Figure 5—figure supplement 1.

Competing interests

The authors declare no competing interests.

References

1. Merrow, M., Franchi, L., Dragovic, Z., Gorl, M., Johnson, J., Brunner, M., Macino, G., and Roenneberg, T., Circadian regulation of the light input pathway in Neurospora crassa. EMBO J, 2001(20): 307-315
2. Vogel, H.J., Distribution of lysine pathways among fungi: evolutionary implications. Am. Naturalist, 1964(98): p. 435-446.