IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/09/02

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(Autocreate 2013/09/02 Entry for IGEM:University_of_East_Anglia_(UEA),_Norwich,_UK/2009/Notebook/NRP-UEA-Norwich_iGEM)
Current revision (11:44, 29 September 2013) (view source)
(Floor One)
 
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==Entry title==
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==Floor One==
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[[Image:GEL3.png|thumb| Fig 1. Gel showing another GUS PCR, with a 1Kb+ base ladder in lane 2 and a band of GUS product at roughly 1.8Kb in lane 3.]]
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The PCR of the new GUS gene was performed (8 x 20 ul reactions of same proportions as first time round).<br>
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A 1% agarose gel was made.<br>
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Took photographs of all Bioassay plates where actinomycete colonies appeared to have a zone of clearing around them - indicating the production of an anti-fungal.<br>
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Spore stocks were filtered, concentrated and resuspended in 20 % glycerol.<br>
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Isolated Streptomyces sp. colonies from the latest soil sample plates and streaked onto new plates. <br>
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Performed some streak purifications and lawns.<br>
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Made 2 x 500 ml SFM (+ Ny).<br>
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An agarose gel electrophoresis of the PCR products showed a fragment at approx. 1.8 Kb which is likely to be the GUS gene ''fig 1''.<br>
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A PCR purification was performed - the product was spun down with PB and PE buffers. The nanodrop indicated that tbere was sufficient DNA to work with. <br>
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A restriction digest was performed on pMS82 and the new GUS gene using NdeI and HindIII enzymes in Buffer B. For the GUS gene digest - 5 ul Buffer, 0.5 ul E1 (enzyme 1), 0.5 ul E2, 35 ul template and 9 ul sterile water was used. For the pMS82 digest - 5 ul Buffer, 0.5 ul E1, 0.5 ul E2, 15 ul template and 29 ul sterile water was used.
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==Floor Two==
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The plates that had been transformed with the ligation (J04450+neo) contained no colonies.

Current revision

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Floor One

Fig 1. Gel showing another GUS PCR, with a 1Kb+ base ladder in lane 2 and a band of GUS product at roughly 1.8Kb in lane 3.
Fig 1. Gel showing another GUS PCR, with a 1Kb+ base ladder in lane 2 and a band of GUS product at roughly 1.8Kb in lane 3.

The PCR of the new GUS gene was performed (8 x 20 ul reactions of same proportions as first time round).
A 1% agarose gel was made.
Took photographs of all Bioassay plates where actinomycete colonies appeared to have a zone of clearing around them - indicating the production of an anti-fungal.
Spore stocks were filtered, concentrated and resuspended in 20 % glycerol.
Isolated Streptomyces sp. colonies from the latest soil sample plates and streaked onto new plates.
Performed some streak purifications and lawns.
Made 2 x 500 ml SFM (+ Ny).
An agarose gel electrophoresis of the PCR products showed a fragment at approx. 1.8 Kb which is likely to be the GUS gene fig 1.
A PCR purification was performed - the product was spun down with PB and PE buffers. The nanodrop indicated that tbere was sufficient DNA to work with.
A restriction digest was performed on pMS82 and the new GUS gene using NdeI and HindIII enzymes in Buffer B. For the GUS gene digest - 5 ul Buffer, 0.5 ul E1 (enzyme 1), 0.5 ul E2, 35 ul template and 9 ul sterile water was used. For the pMS82 digest - 5 ul Buffer, 0.5 ul E1, 0.5 ul E2, 15 ul template and 29 ul sterile water was used.

Floor Two

The plates that had been transformed with the ligation (J04450+neo) contained no colonies.



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