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23-06-2014 - Streaking wt. B.subtilis on LBA

AIM: To obtain single colonies of Wt b.Subtilis

We received the wt (1A1) and mutant BKE24040, BKE24050, BKE24080 from BGSC on 23-06-2014. The wt strain (1A1) was on a paper strip. We received 3 strips from BGSC. We plated two strips in two different LBA plates. The plates were free from antibiotics. We placed the strips gently on the LBA and added 1 drop of sterile H2O. After few seconds we streaked the paper strip on the LBA and discarded the strip. The LBA plates were incubated at 37° C

 

 

24-06-2014 - Re-Streaking wt. B.subtilis on LBA to obtain single colonies

AIM: To obtain single colonies of Wt b.Subtilis from 23-06-14 plate

we took a single colony and streaked it on a new LBA with and w/o Erythromycin.

Observation:

No colonies in LBA + EM
Colonies observed in LBA w/o EM

 

 

27-06-2014 - Preparing LBA and LB broth with Erythromycin

AIM: To prepare LBA and LB broth for B.Subtilis mutants

We added 2.5 ul of the EM (20mg/ml) to 50ml of LB broth. We added 25 ul of EM to 250 ml of LBA and prepared the plates & stored them at 4° C.

 

 

30-06-14 - Preparation of miniculture of wild type and mutant B.Subtilis for glycerol stock

AIM: To prepare 10% glycerol stock of wt and mutant (BKE24040, BKE24050, BKE24080) B.Subtilis

We inoculated  Single colony of both wt (1A1) and mutant (BKE24040, BKE24050, BKE24080) in 5ml of LB broth supplemented w/o EM for wt and with EM for mutants. Incubated the colonies @ 37°C overnight.

After the overnight incubation the tubes were stirred well. 750 ul of the culture is transferred to cryogenic tube and 750 ul of 60% glycerol is added to the culture to obtain 10% glycerol stock.

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