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

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Autocreate 2013/08/05 Entry for IGEM:University_of_East_Anglia_(UEA),_Norwich,_UK/2009/Notebook/NRP-UEA-Norwich_iGEM)
Line 5: Line 5:
|-
|-
| colspan="2"|
| colspan="2"|
-
==Entry title==
+
==Floor One==
-
* Insert your content here.
+
A total of 49 liquid cultures from streak-purified single colony Streptomyces plates were prepared previously (DATE?) 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.
 +
 
 +
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.

Revision as of 12:15, 15 August 2013

iGEM Project name 1 Main project page
Previous entry      Next entry

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.

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.

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.



Personal tools