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Team:DTU-Denmark/Notebook/18 July 2013
From 2013.igem.org
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(pZA21 amplified with USER primers and DpnI treated) | (pZA21 amplified with USER primers and DpnI treated) | ||
| - | Per one reaction we | + | Per one reaction we mixed 3uL of USER mix + 7uL of insert |
Inserts: AMO, HAO, water (negative control) | Inserts: AMO, HAO, water (negative control) | ||
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Transformation of chemically competent ''E. coli'' cells. | Transformation of chemically competent ''E. coli'' cells. | ||
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| + | 10uL of USER construct after USER reaction was added to 100uL of competent cells. Left on ice for 30 min, heat shock for 90 sec at 42 C and left on ice for 2-5 min. Added SOC medium, incubated at 37C with shaking for 2 hours. | ||
| + | Plated on plates with kanamycin. | ||
| + | Left overnight at 37C to let transformants grow. | ||
Revision as of 15:46, 18 July 2013
Contents |
208
Main purpose
- USER reaction
Who was in the lab
Gosia, Henrike, Julia
Procedure
- made gel for yesterday's PCR of Nir operon -> empty
- USER reaction with HAO + pZA21 and AMO + pZA21
- nandrop measurement of yesterday's miniprep showed it had very small yield, so we will redo it
- inocculated cultures for miniprep of RFP in pZA21 and cycAX in pZA21
Gel electrophoresis - Nir, cycAX and plasmids from (3 with RFP, one with cycAX) miniprep
Gel electrophoresis parameters: 1% agarose gel, 80V, 55 min
USER reaction
Was performed by standard procedure
USER mix contained (per 1 sample):
- USER enzyme - 1uL
- NEB buffer 4 - 0.5uL
- 10x BSA - 0.5uL
- backbone pZA21 - 1 uL
(pZA21 amplified with USER primers and DpnI treated)
Per one reaction we mixed 3uL of USER mix + 7uL of insert Inserts: AMO, HAO, water (negative control)
Incubation - USER reaction 40 min at 37 C 30 min at 25 C
Transformation of chemically competent E. coli cells.
10uL of USER construct after USER reaction was added to 100uL of competent cells. Left on ice for 30 min, heat shock for 90 sec at 42 C and left on ice for 2-5 min. Added SOC medium, incubated at 37C with shaking for 2 hours. Plated on plates with kanamycin. Left overnight at 37C to let transformants grow.
