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Team:DTU-Denmark/Notebook/18 July 2013
From 2013.igem.org
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* inocculated cultures for miniprep of RFP in pZA21 and cycAX in pZA21 | * inocculated cultures for miniprep of RFP in pZA21 and cycAX in pZA21 | ||
| - | === Gel electrophoresis - Nir, | + | === 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 mix 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. | ||
Revision as of 15:42, 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 mix 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.
