Difference between revisions of "IGEM:IMPERIAL/2006/LabCalendar/2006-9-7"

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(Miniprep & Maxiprep)
(Ligation & electroporation)
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==Ligation & electroporation==
==Ligation & electroporation==
* New J37023 (Flagged and tagged Aiia from Dr Fray) insert ligated into previously maxipreped stock of 1I (B0015).
* New J37023 (Flagged and tagged Aiia from Dr Fray) insert ligated into previously maxipreped stock of 1I (B0015).
*Part J37024
*Electroporated at 5.15pm.
*Electroporated at 5.15pm.

Latest revision as of 02:11, 8 September 2006

*Electroporate Dr. Fray's plasmid containing aiiA into DH5a 
 If any colonies grow until evening - culture up for testing on Friday !!
*Maxi & mini of J37022, aiiA (with flag tag), aiiA
*Next ligation step for new predator cell (with the new aiiA) once maxiprep is finished & electroporation on same day ! 
*Testing of J37015, J37022, Acylace Activity (need J37016 for two of them)
*Send J37022 for sequencing
*Make frozen stock of new T9002, J37036, J37022 !!!
*Control digest for J37036 - Has ligation worked?

*JS: Also, you guys need to start sending in parts to the registry, or else we won't have anything to show for all our efforts this summer!

*JW: Sorted out the fridge today - we have all the maxis, ready to be sent when necessary, however we have multiple copies of some. We need to determine which is the one that was used in construction.


  • The new aiiA was sent to us in DH5-α. However the two promoters in the pGEM cloning vector in which it was sent (T7 and Sp6) do not work in DH5-α cells, and so attempting to test for expression here would be pointless
  • We can however test for expression in Rosetta, which can use the T7 promoter.

Miniprep & Maxiprep

  • Are the results successful?
  • Maxiprep:
    • All four J37022 cultures successfully maxipreped
    • One of the two Aiia cultures successfully maxipreped. DNA used for ligations

Ligation & electroporation

  • New J37023 (Flagged and tagged Aiia from Dr Fray) insert ligated into previously maxipreped stock of 1I (B0015).
  • Part J37024
  • Electroporated at 5.15pm.

Electroporation of Dr. Fray's plasmid containing aiiA

  • Was electroporated this morning into Rosetta (not into DH5a because no expression will take place in DH5a due to the promoter not working in this strain)
  • Might get some colonies so that it can be cultured for Western Blot tomorrow

Testing J37015

  • Testing was carried out partly according to the current J37015 protocol and partly with some added measurements and amended steps. These are outlined below. (The protocol will be adjusted accordingly)
  • Put into 2hrs shake at 9:45
  • Taken out of shaker at 12:00
  • Measure OD: 1.099 and dilute into 25mL to OD 0.1
  • Spin down the cells & discard supernatant
  • Add 25mL of prewarmed LB & resuspend
  • Made up 2 x 25mL cultures
    • One is inoculated with 1nM AHL (add 5μL of 5uM stock to the 25 mL)
    • The other one is not inoculated with AHL

In Dr. Mann's Lab:

  • Place in shaker
  • Two samples were taken every 10-15min. for 2hrs
    • One sample: Used Wallac-Victor III after taking samples to read fluorescence and OD (in order to detect GFP levels)
    • Second sample: Supernatant is stored in order to assess AHL levels with J37016 later

Assessing AHL of samples with J37016:

  • Replaced supernatant of J37016 with saved supernatant from above
  • Put into shaker for 4hrs shake at 16:00
  • Taken out of shaker at 19:30
  • Fluorescence reading to assess amounts of AHL present (using J37016) afterwards

Acylase Check

This was done to check whether acylase degrades anything that is vital to DH5a

  • Put 2 x 2mL J37016 (OD 0.1) in shaker at 16:20
    • One of them is inoculated with 8μL of Soln 1 of the acylase
    • No enzyme was added to the other culture (control)
  • OD will be checked & compared after a few hours in shaker

Control digest of J37036

  • Identified enzymes - will be run first thing tomorrow morning


  • Smaller fragment worked.....at last and so we will give the PCR fusion a go tomorrow