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Team:TUDelft/14 July 2009
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Prepared 1 litre of LB agar medium and sent to Kluyver lab's kitchen. | Prepared 1 litre of LB agar medium and sent to Kluyver lab's kitchen. | ||
| - | Using 650 | + | Using 650 µl of competent cells (E.coli DH5alpha) and the transformation protocol, we inserted 13 biobricks [R0010, I14018, I12006, R0040, J23008, J23031, E0040, E1010, C0051, B0034, B0015, S03335, S03473, K081013 and C0040] for delay into competent cells and grew them in a solid media to increase the amount of biobricks we will have. |
| - | Diluted the 13 biobricks with 15 | + | Diluted the 13 biobricks with 15 µl RNase free water and stored in -20°C freezer. |
Standard transformation procedure: | Standard transformation procedure: | ||
| - | Remove competent cells from -80, let thaw for 10 min on ice and aliquot in 50 | + | Remove competent cells from -80°, let thaw for 10 min on ice and aliquot in 50 µl amounts.<br> |
| - | add 2-5 | + | add 2-5 µl of vector, to 50 µl cells, no mixing by pipet due to shear induction.<br> |
keep on ice for 20 minutes (vector spreading through volume).<br> | keep on ice for 20 minutes (vector spreading through volume).<br> | ||
heat shock (42°C) for 45 seconds.<br> | heat shock (42°C) for 45 seconds.<br> | ||
keep on ice for 2 minutes.<br> | keep on ice for 2 minutes.<br> | ||
| - | add 200 | + | add 200 µl SOC, put on 37°C for 1 hour or longer with agitation (160 rpm).<br> |
| - | plate out 250 | + | plate out 250 µl on appropriate antibiotics.<br> |
=='''Weenink'''== | =='''Weenink'''== | ||
Latest revision as of 14:30, 25 July 2009
Lab Notebook
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14th July
Lab work: Sriram & Orr
Prepared 1 litre of LB agar medium and sent to Kluyver lab's kitchen.
Using 650 µl of competent cells (E.coli DH5alpha) and the transformation protocol, we inserted 13 biobricks [R0010, I14018, I12006, R0040, J23008, J23031, E0040, E1010, C0051, B0034, B0015, S03335, S03473, K081013 and C0040] for delay into competent cells and grew them in a solid media to increase the amount of biobricks we will have.
Diluted the 13 biobricks with 15 µl RNase free water and stored in -20°C freezer.
Standard transformation procedure:
Remove competent cells from -80°, let thaw for 10 min on ice and aliquot in 50 µl amounts.
add 2-5 µl of vector, to 50 µl cells, no mixing by pipet due to shear induction.
keep on ice for 20 minutes (vector spreading through volume).
heat shock (42°C) for 45 seconds.
keep on ice for 2 minutes.
add 200 µl SOC, put on 37°C for 1 hour or longer with agitation (160 rpm).
plate out 250 µl on appropriate antibiotics.
Weenink
Brought beaker of eppendorf tubes to autoclaving
Calin
Looked into the details of lambda red knockout protocol. Started designing the required primers.
