IGEM:IMPERIAL/2006/LabCalendar/2006-9-7

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(Miniprep & Maxiprep)
Current revision (05:11, 8 September 2006) (view source)
(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.

Current revision



TO DO:
*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.

Contents

aiiA

  • 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

LoxP PCR

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