Team:Goettingen/Experiments



LB Medium

Plasmid Extraction - using peqGOLD Plasmid Miniprep Kit I (PEQLAB Technologies)

Plasmid Extraction - using QIAprep Spin Miniprep Kit (QIAGEN)

Blunt End Ligation in pJET1.2 vector –Clone JET PCR Cloning Kit–Thermo Scientific

TOPO® Cloning protocol usingChampion™ pET Directional TOPO® Expression Kits. Thermo Fisher Scientific

PCR product purification using QIAquick® PCR Purification Kit (QIAGEN)

Plasmid transformation into chemically competent E. coli

Preparation of competent E.coli cells

Esterase activity test

PCR Gel extraction, peqGOLD Gel Extraction Kit

Electroporation of BL21 cells with pJET_RFP

Media preparation for cellulase activity screening

Cellulase activity screening

 

Esterase Activity plates, with 1% Tributyrin

 

To 500ml of LB Media add 7,5g of Agar and 5ml of Tributyrin and homogenize with a mixer.

This culture medium must be directly sterilized by autoclaving at 121˚C for 20 min.

If you wait too long it will be inhomogeneous again!

When the medium cools down enough, the antibiotic can be added.

 

Result: Halo formation is visible around the positive clones.

 

Phosphatase Activity plates, Sperber media

 

A: Stock reagents

1M IPTG (4,76 g in 20ml Millipore-H2O) filter with blue 0,22 um sterile filter and freeze at -20˚C

50mg/ml Kanamycin in Millipore H2O, filter with blue 0,22 um sterile filter and freeze at -20˚C

100mg/ml Ampicillin in 50% ethanol, filter with blue 0,22 um sterile filter and freeze at -20˚C

25mg/ml BCIP (in DMF) filter with blue 0,22 um sterile filter and keep in a falcon tube wrapped with aluminum foil at 4˚C. BCIP: Biomol Nr. 2291 (MG 433,64)

Glycerol (99%), autoclaved and kept at room temperature

 

B: Sperber medium

1g Yeast extract

3,5 ml 50% w/w Phytic acid

0,2g CaCl2

0,5g MgSO4

Adjust the pH to 7,2 with NaOH

Ad 2L of Millipore H2O

32g agar

Autoclave

 

After autoclaving the medium must cool down to ca. 50˚C. Now glycerol, IPTG, BCIP and the respective antiobiotic can be added.

 

For 2 L medium:

2ml BCIP

2ml 1M IPTG

2ml Ampicllin or Kanamycin

40mL Glycerol

The media is now ready for plating


Result: On Sperber medium phosphatase-recombinant colonies should develop a distinct color blue after 2 days.

 

Experiments & Protocols

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