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.