Team:Cambridge/Notebook/Week4

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== Monday ==
== Monday ==
 +
===Wet Work===
 +
 +
====Melanin====
 +
 +
*Results from weekend plates:
 +
 +
<need a picture>
 +
 +
It appears that the bacteria were much too high a concentration; they formed lawns.  Apparently pigment production is reduced at high bacterial concentrations.  However, it is encouraging that the darkest plate is the one with the greatest tyrosine concentration and IPTG.
 +
 +
To follow up, the experiment was repeated with 1/100 and 1/1000 dilutions of bacterial culture on the following plates:
 +
 +
100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.3 mg/mL tyrosine
 +
100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.6 mg/mL tyrosine
 +
100ug/ml Ampicillin, 7.5 ug/ml CuSO4, 0.075 mg/mL tyrosine, 0.5mM IPTG
 +
100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.3 mg/mL tyrosine, 1mM IPTG
 +
100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.6 mg/mL tyrosine, 1mM IPTG
 +
 +
*Unfortunately, the overnight cultures for the plate readers were contaminated, so a single colony was once again incubated overnight in 10 mL of 100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.3 mg/mL tyrosine, 1mM IPTG  in preparation for pigment characterisation using the plate reader.
 +
 +
====Ampiflication====
 +
 +
*Chose 5 activator / promoter combinations to continue work with:
 +
:*P2 ogr activator with
 +
::*PO promoter (I1746371)
 +
::*Psid promoter (I746374)
 +
:*phiR73 delta activator with
 +
::*PF promoter (I746390)
 +
::* PO promoter (I746391)
 +
::* Psid promoter (I74394)
 +
 +
*Of the above, on plates all showed leaky expression of GFP and RFP, suggesting that last weeks transformation was successful.
 +
 +
*Inoculated 5 replicates of single colonies overnight of each in preparation for colony PCR and miniprep tomorrow.
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Revision as of 10:45, 3 August 2009


Week 1

Monday

Wet Work

Melanin

  • Results from weekend plates:

<need a picture>

It appears that the bacteria were much too high a concentration; they formed lawns. Apparently pigment production is reduced at high bacterial concentrations. However, it is encouraging that the darkest plate is the one with the greatest tyrosine concentration and IPTG.

To follow up, the experiment was repeated with 1/100 and 1/1000 dilutions of bacterial culture on the following plates:

100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.3 mg/mL tyrosine 100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.6 mg/mL tyrosine 100ug/ml Ampicillin, 7.5 ug/ml CuSO4, 0.075 mg/mL tyrosine, 0.5mM IPTG 100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.3 mg/mL tyrosine, 1mM IPTG 100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.6 mg/mL tyrosine, 1mM IPTG

  • Unfortunately, the overnight cultures for the plate readers were contaminated, so a single colony was once again incubated overnight in 10 mL of 100ug/ml Ampicillin, 15 ug/ml CuSO4, 0.3 mg/mL tyrosine, 1mM IPTG in preparation for pigment characterisation using the plate reader.

Ampiflication

  • Chose 5 activator / promoter combinations to continue work with:
  • P2 ogr activator with
  • PO promoter (I1746371)
  • Psid promoter (I746374)
  • phiR73 delta activator with
  • PF promoter (I746390)
  • PO promoter (I746391)
  • Psid promoter (I74394)
  • Of the above, on plates all showed leaky expression of GFP and RFP, suggesting that last weeks transformation was successful.
  • Inoculated 5 replicates of single colonies overnight of each in preparation for colony PCR and miniprep tomorrow.
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