IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/05: Difference between revisions

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==Floor One==
==Floor One==
A total of 49 liquid cultures from streak-purified single colony Streptomyces plates were prepared previously (DATE?) through inoculation in LB broth with springs.
A total of 49 liquid cultures from streak-purified single colony ''Streptomyces'' plates were prepared previously through inoculation in LB broth with springs.


Made up 30 mg/ml lysozyme - 10 ml distilled water and 300 mg of lysozyme. The mix was filter sterilised and 10 ul added to each eppendorf of liquid culture (49). The liquid cultures were then centrifuged to remove as much supernatant as possible. A 400 ul aliquot of Buffer 1 (Tris and EDTA) was added to each culture containing the lysozyme. The eppendorfs were incubated at 37 °C for 1 hr to allow the lysis reaction to proceed.
Made up 30 mg/ml lysozyme stock. The mix was filter sterilised and 10 ul added to each eppendorf of liquid culture (49). The liquid cultures were then centrifuged to remove as much supernatant as possible. A 400 ul aliquot of Buffer 1 (TE buffer containing 10mM Tris and 1mM EDTA) was added to each culture containing the lysozyme. The eppendorfs were incubated at 37 °C for 1 hr to allow the lysis reaction to proceed.


PCR was then performed on the 49 lysed cultures. A Master Mix was prepared - 110 ul Buffer, 55 ul DMSO, 22 ul dNTPs, 55 ul Primer 1, 55 ul Primer 2, 33 ul MgCl2, 742.5 ul dH20, 11 ul Taq polymerase. 19.6 ul of Master Mix was added to 0.4 ul of lysed culture (template DNA). The mixes were placed in a PCR machine for a 3 hr cycle.  
PCR was then performed on the 49 lysed cultures. A Master Mix was prepared - 110 ul Buffer, 55 ul DMSO, 22 ul dNTPs, 55 ul Primer 1, 55 ul Primer 2, 33 ul MgCl2, 742.5 ul dH20, 11 ul Taq polymerase. 19.6 ul of Master Mix was added to 0.4 ul of lysed culture (template DNA). The mixes were placed in a PCR machine for a 3 hr cycle.  

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Floor One

A total of 49 liquid cultures from streak-purified single colony Streptomyces plates were prepared previously through inoculation in LB broth with springs.

Made up 30 mg/ml lysozyme stock. The mix was filter sterilised and 10 ul added to each eppendorf of liquid culture (49). The liquid cultures were then centrifuged to remove as much supernatant as possible. A 400 ul aliquot of Buffer 1 (TE buffer containing 10mM Tris and 1mM EDTA) was added to each culture containing the lysozyme. The eppendorfs were incubated at 37 °C for 1 hr to allow the lysis reaction to proceed.

PCR was then performed on the 49 lysed cultures. A Master Mix was prepared - 110 ul Buffer, 55 ul DMSO, 22 ul dNTPs, 55 ul Primer 1, 55 ul Primer 2, 33 ul MgCl2, 742.5 ul dH20, 11 ul Taq polymerase. 19.6 ul of Master Mix was added to 0.4 ul of lysed culture (template DNA). The mixes were placed in a PCR machine for a 3 hr cycle.

Made 4 x 500 ml SFM agar media, autoclaved for 1 hr. Added 5 ml MgCl2 to one flask, 500 ul Nystatin to 3 flasks and poured into plates.



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