IGEM:IMPERIAL/2007/Notebook/2007-9-3

name=iGEM:IMPERIAL/2007/Notebook date=2007/09/24 view=threemonths format=%name/%year-%month-%day weekstart=7

Construction of pT7-GFP

 * 1) Minipreped the 5 overnight cultures

Protocols can be found at Miniprep in the general protocols page


 * 1) Digested 5μl of DNA with Nde1 and BamH1
 * 2) Checked the digestion on 1% agarose gel
 * 3) *1. Colony 1
 * 4) *2. Colony 1 digested
 * 5) *3. Colony 2
 * 6) *4. Colony 2 digested
 * 7) *5. Colony 3
 * 8) *6. Colony 3 digested
 * 9) *7. Colony 4
 * 10) *8. Colony 4 digested
 * 11) *9. Colony 5
 * 12) *10. Colony 5 digested
 * 13) Colonies 1 and 4 show the correct-size insert upon digestion



Protocols can be found at Electrophoresis in the general protocols page


 * 1) Transformed recombinant plasmid DNA into BL21
 * 2) Plated transformed cells onto LB + Kan plates
 * 3) Left plates at 37&deg;C overnight

Protocols can be found at Transformation in the general protocols page

Maxiprep of Biobricks

 * 1) Maxipreped 2 Biobricks


 * 2. BBa_I13422 [ptet-GFP]
 * 18. BBa_T9002 [plux-GFP]

Protocols can be found at Maxiprep in the general protocols page



Conclusions:
 * Part 2 is very concentrated!
 * Part 27 and 29 are not cloned properly

Vesicles
Formation of Vesicles

For both the POPC/dodecane 10ml and DOPC/dodecane 10ml suspensions prepared today - without overnight incubation - the following steps were taken:
 * 2ml of suspension was taken to prepare an interface according to protocol.
 * 25&mu;l of 100x diluted GFP solution was used to prepare the emulsion; stirred gently with magnetic stirrer.

'''NOTE: There was a mistake in labelling the containers for the two suspensions - both were mistakenly labelled as DOPC. When the mistake was spotted, it was impossible to distinguish between the POPC and DOPC containers. The labels used then were a best guess, and have been carried through the remainder of these results. So, please bear in mind that the results given could either be POPC or DOPC.'''

4 samples prepared:
 * Sample 1: 1ml of POPC/dodecane/GFP emulsion added over interface prepared with 2ml of POPC/dodecane suspension.
 * Sample 2: 1ml of DOPC/dodecane/GFP emulsion added over interface prepared with 2ml of DOPC/dodecane suspension.
 * Sample 3: 2ml of POPC/dodecane/GFP emulsion, with no previously prepared interface.
 * Sample 4: 2ml of DOPC/dodecane/GFP emulsion, with no previously prepared interface.

Samples 1 and 2 were centrifuged at 120x g. Samples 3 and 4 have been left overnight for sedimentation to occur.

Results Samples 1 and 2 were collected for observation under the microscope. The following results were obtained:
 * Sample 1: Vesicles observed, but not photographed.
 * Sample 2: Vesicles observed.

Preparations

Three experiments were set up for tomorrow:


 * 1) One 100ml beaker with 250&mu;l of POPC and 50ml of dodecane
 * 2) One 50mm by 25mm cylinder with 50&mu;l of POPC and 10ml of dodecane
 * 3) One 50mm by 25mm cylinder with 50&mu;l of DOPC and 10ml of dodecane

All desiccated and sonicated, and left on the bench overnight (not the incubator).