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Lab 208

Main purpose

• USER reaction and transformation

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

• dilution of new primers which arrived today

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.

Primers dilution

Primers arrived in dried form. We diluted it in 150uL of milliQ water (diluted 150 times) and saved as stock at -80C. From stock we took 10uL and added to 90uL of water (diluted 10 times) to make working solution of primers kept in -20C.

PCR to insert his-tags into the hello world constructs

Set up new PCR reaction for this purpose, using two different ramp programs.

ramp-1: from 60C to 50C in steps of 0.1C/sec

ramp-1: from 60C to 55C in steps of 0.1C/sec

Elongation time 4:00 for both programms

Conclusion

Gel photo didn't show expected Nir fragment and cycAX.

Nanodrop measurements and gel photo showed very low yield in miniprep. LB medium with kanamycin was inoculated again and tomorrow we will perform plasmid isolation using Qiagen kit.

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