Melbourne/Lab Notebook gv F3

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< Melbourne(Difference between revisions)
 
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*Leave at room temperature and observe the settling over the next week.
*Leave at room temperature and observe the settling over the next week.
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==Example pNL29(orininal plasmid) and Tube(64) the IPTG promoted version of BBa_I750016==
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==Floatation test of mutated cell lines in DH5a cells==
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In the following series of photos the tubes are in pairs. The outer pairs are uninduced (no IPTG), the inner pairs are induced. The tubes left of centre are the original pNL29 plasmid in DH5a cells, the tubes on the right are 41C plasmids (promoted BBa_I750016) in XL10 gold. We believe the XL10-gold is permanenently inducing the expression even without IPTG, this seems to be confirmed by the latter experiment where the plasmid was retransformed into DH5alpha cell.  
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In the following series of photos the tubes are in pairs. The left hand tube in each pair is uninduced (no IPTG), the right hand tube in each pair is induced with 1mM IPTG. The left pair of tubes is the original pNL29 plasmid in DH5a cells, the next pair of tubes on the right are 41C plasmids (promoted BBa_I750016) in DH5a. The other tubes are the results of the parrellel development path 42 & 43 are quad mutated much as 41C is, 44 is a quad mutated version of pNL26 allowing the three gas vesicle genes not in pNL29/41C to be converted to biobricks.
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[[Image:Melbourne-float1 6pm 17-10-2007.jpg|850px|thumb|6hrs (6pm 17-10-2007)]]
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[[Image:Melbourne-float2 11Am 22-10-2007.jpg|850px|thumb|In LB  (11Am 22-10-2007)]]
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[[Image:Melbourne-float1 11am 18-10-2007.JPG|850px|thumb|11hrs (11Am 18-10-2007)]]
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[[Image:Melbourne-float2 12pm 22-10-2007.jpg|850px|thumb|In saline 0hrs (12pm 22-10-2007)]]
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[[Image:Melbourne-float1 6pm 20-10-2007.jpg|850px|thumb|78hrs (6pm 20-10-2007)]]
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[[Image:Melbourne-float2 1pm 23-10-2007.jpg|850px|thumb|25hrs (1pm 23-10-2007)]]
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[[Image:Melbourne-float1 4pm 21-10-2007.jpg|850px|thumb|100hrs (4pm 21-10-2007)]]
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[[Image:Melbourne-float 2 3pm 24-10-2006.jpg|850px|thumb|51hrs (3pm 24-10-2007)]]
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[[Image:Melbourne-float2 3pm 25-10-2007.jpg|850px|thumb|75hrs (3pm 25-10-2007)]]
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[[Image:Melbourne-float2 12pm 26-10-2007.jpg|850px|thumb|96hrs (12pm 26-10-2007)]]

Latest revision as of 14:22, 26 October 2007

<Return to lab book summary>

Testing Floatation

  • Growing up cells from glycerol stocks overnight in 5mL LB amp
  • Prepare 250ml conical shaker flasks by cleaning and autoclave.
  • Add 50mL of LB Amp to each of two flasks.
  • Add 1M IPTG to one flask to make final concentration of 1mM IPTG, Lable with I.
  • Transfer 1mL of overnight culture to each flask.
  • Grow overnight at 37degC on a shaker.
  • Next morning transfer to 50ml falcon tubes.
  • Spin down the cells gently 25minutes at 1000rpm in Eppendorph 5810R centrifuge.
  • Poor off and discard the LB, maintaining the pellet and a residual ammount of LB 1ml about.
  • Replace LB with 50ml of 10g/L NaCl filtered through a 0.22um filter.
  • Resuspend the cells by shaking.
  • Leave at room temperature and observe the settling over the next week.

Floatation test of mutated cell lines in DH5a cells

In the following series of photos the tubes are in pairs. The left hand tube in each pair is uninduced (no IPTG), the right hand tube in each pair is induced with 1mM IPTG. The left pair of tubes is the original pNL29 plasmid in DH5a cells, the next pair of tubes on the right are 41C plasmids (promoted BBa_I750016) in DH5a. The other tubes are the results of the parrellel development path 42 & 43 are quad mutated much as 41C is, 44 is a quad mutated version of pNL26 allowing the three gas vesicle genes not in pNL29/41C to be converted to biobricks.

In LB (11Am 22-10-2007)
In saline 0hrs (12pm 22-10-2007)
25hrs (1pm 23-10-2007)
51hrs (3pm 24-10-2007)
75hrs (3pm 25-10-2007)
96hrs (12pm 26-10-2007)
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