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Team:TUDelft/14 July 2009
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=='''Lab work: Sriram & Orr'''== | =='''Lab work: Sriram & Orr'''== | ||
| - | Using 650 ul of competent cells and the transformation protocol, we inserted 13 biobricks for delay into those competent cells and grew them in a solid media to increase the amount of biobricks we will have. | + | Using 650 ul of competent cells (E.coli DH5alpha) and the transformation protocol, we inserted 13 biobricks for delay into those competent cells and grew them in a solid media to increase the amount of biobricks we will have. |
Diluted the 13 biobricks with 15 ul RNase free water and stored in -20°C freezer. | Diluted the 13 biobricks with 15 ul RNase free water and stored in -20°C freezer. | ||
Revision as of 10:47, 15 July 2009
14th July
Lab work: Sriram & Orr
Using 650 ul of competent cells (E.coli DH5alpha) and the transformation protocol, we inserted 13 biobricks for delay into those competent cells and grew them in a solid media to increase the amount of biobricks we will have.
Diluted the 13 biobricks with 15 ul 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 ul amounts.
add 2-5 ul of vector, usually in H2O, to 50 ul 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 ul SOC, put on 37°C for 1 hour or longer with agitation (160 rpm).
plate out 250 ul on appropriate antibiotics.
Prepared 1 litre of LB agar medium and sent to Kluyver lab's kitchen.
Weenink
Brought beaker of eppendorf tubes to autoclaving
