Imperial College/17 August 2008
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- | = | + | =17 August 2008= |
==Wet Lab== | ==Wet Lab== | ||
===''B.subtilis=== | ===''B.subtilis=== | ||
+ | =====Monday===== | ||
+ | *Prepare media and reagents for the protocol transformation 1 protocol | ||
+ | *Prepare 2x10ml LB media in 100ml flasks | ||
+ | *Prepare 2x100ml LB media in 1000ml flasks | ||
+ | *Prepare 2x overnight cultures in the 10ml LB media flasks | ||
+ | *Prepare a 1% agarose gel and run the digest prepared on friday | ||
+ | |||
+ | =====Tuesday===== | ||
+ | *Prepare competent cells using the transformation 2 protocol, this time growing cells to an o.D.<sub>600</sub> | ||
+ | *Prepare 1x20ml of LB media in a 200ml flask (autoclaved in the morning) | ||
+ | *Prepare 1x20ml of SpC media in a 200ml flask (autoclaved in the morning) | ||
+ | *Prepare aliquots of SpII media (autoclaved in the morning) | ||
+ | |||
+ | =====Wednesday===== | ||
+ | *Prepare competent cells using the transformation 1 protocol, | ||
+ | *Electroporate the compete cells from the transformation protocol 2 using a range of concentrations | ||
+ | |||
+ | =====Thursday===== | ||
+ | *Transform the competent cells prepared from transformation 2 protocol | ||
+ | *Check the transformation cells from protocol 2, if transformed correctly then carry the test for correct integration, | ||
+ | |||
+ | =====Friday===== | ||
+ | *If the transformation has been successful then carry out the integration test. | ||
===Cloning=== | ===Cloning=== | ||
+ | |||
=====Monday===== | =====Monday===== | ||
- | Prepare plates and Media for later in the week | + | *Prepare plates and Media for later in the week |
- | Transform ''E.coli'' Xl1-blue with C0012 (LacI) from the registry again | + | *Transform ''E.coli'' Xl1-blue with C0012 (LacI) from the registry again |
=====Tuesday===== | =====Tuesday===== | ||
- | Check LacI transformants | + | *Check LacI transformants |
- | Miniprep and digest 0.1.01 (Double Terminator) and 0.1.02 (Chloraphenicol Reisitance) | + | *Miniprep and digest 0.1.01 (Double Terminator) and 0.1.02 (Chloraphenicol Reisitance). |
+ | **Ligate together to form construct 0.1.14 and transform into Xl1-Blue ''E.coli'' | ||
=====Wednesday===== | =====Wednesday===== | ||
- | Check ''E.coli'' transformed with construct 0.1.14 for growth and by PCR (if growth was succesful) | + | *Check ''E.coli'' transformed with construct 0.1.14 for growth and by PCR (if growth was succesful) |
- | If primers have arrived, PCR clone LacI, XylR, Spectinomycin, EpsE modified integration sequnce and AmyE integration sequence into Biobricks | + | *If primers have arrived, PCR clone LacI, XylR, Spectinomycin, EpsE modified integration sequnce and AmyE integration sequence into Biobricks |
=====Thursday===== | =====Thursday===== | ||
- | If grown, mini/midi-prep a few colonies from each plate and digest a sample with ''XbaI'' and ''SpeI'' to | + | *If grown, mini/midi-prep a few colonies from each plate and digest a sample with ''XbaI'' and ''SpeI'' to determine which plasmids have the insert in the correct orientation. |
- | If primers arrive today PCR cloning should be carried out | + | *If primers arrive today PCR cloning should be carried out |
=====Friday===== | =====Friday===== | ||
- | + | *If primers arrived Thursday check plates for growth and carry out minipreps | |
+ | |||
+ | *PCR check the minipreps that could be digested by ''XbaI'' and ''SpeI'' | ||
+ | <br> | ||
+ | {{Imperial/EndPage|Notebook|Notebook}} |
Latest revision as of 20:37, 28 October 2008
17 August 2008Wet LabB.subtilisMonday
Tuesday
Wednesday
Thursday
Friday
CloningMonday
Tuesday
Wednesday
Thursday
Friday
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