IGEM:IMPERIAL/2006/LabCalendar/2006-9-4

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TO DO:
*Testing of T9002, J37016, J37020, S01656, J37015, J37015RS
*Culture J37036 & J37018 from plate in incubator for maxi & mini on Tuesday
*Make frozen stock of J37022 & culture up for testing ! 
*Send J37024 for sequencing !!!
*Order new maxiprep kit !!!
*Need more autoclaved flasks !!!
*Need more isopropanol too!

Contents

Ligations

  • J04500 (vector) and J37024 (insert)being ligated overnight

LoxP PCR

  • Was done for the smaller fragment
  • Success to be determined tomorrow

Western Blot

  • 3 colonies of Rosetta and 3 colonies of BL21 containing S01656 were cultured up.
  • The samples were recultured for a couple of hours to bring them back to an exponential growth phase
  • The samples were pipetted into 2 sets of Eppendorfs, one of which was induced with IPTG
  • The Western blot was run, and the gel was left to stain
  • The results will be known tomorrow morning

Maxiprep

  • Maxiprep new AiiA gene from overnight culture.
  • Not very much bacteria (hence DNA) present.

AiiA PCR

  • Using the new primers and the newly arrived AiiA gene (maxi-preped this morning), PCR carried oput to add the LVA tag and immunotag.
  • PCR annealing temperatures calculated to be 10 cyles at 51[[:Category:{{{1}}}|{{{1}}}]] and 20 cycles at 69[[:Category:{{{1}}}|{{{1}}}]]

Culturing

  • S01656 +IPTG, S01656 -IPTG, J37016 was cultured for testing
  • J37036 & J37018 & T9002 could not be cultured since no colonies could be seen on the plate - they have to grow overnight

Frozen Cells

  • Made a frozen stock of cells: J37025 (3 different colonies, but all of them have been verified to be correct)
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