2019.7.20 Sat
Read the literature
2019.7.21 Sun
Read the literature on arsenic treatment in Bangladesh
2019.7.22 Mon
Upon first entry into the laboratory, arsenic adsorption plasmids had been synthesized by two plasmids, pEP-SpPCS and pET-orpF-fmt, transforming E. coli BL21(DE3) and culture overnight
2019.7.23 Tue
Select a single colony on the plate, convert the colonies of pEP-SpPCS and pET-orpF-fmt, inoculate LB broth containing ampicillin resistance, and culture overnight.
According to the designed primer, J33201fragment was amplified, electrophoresis identification, gel cutting and purification, enzyme cutting E/S, gel cutting and purification
2019.7.24 Wed
The bacterial liquid cultured overnight was re-inoculated into fresh LB broth containing ambenzyl resistance at a ratio of 1:100 for expanded culture and detection of OD600. When OD value was 0.6-0.8, IPTG(1mM) was added for induction expression and low temperature was induced overnight at 28℃.
2019.7.25 Thu
Protein samples were prepared with 5* SDS-PAGE loading buffer
SDS-PAGE gel was prepared and SDS-PAGE electrophoresis was carried out on the prepared samples
After electrophoresis, Coomassie brilliant blue was dyed for 30min, then decolorized and washed overnight
2019.7.26 Fri
SDS-PAGE electrophoresis results were observed and photographed
Prepare HUST communication materials
SDS-PAGE adhesive was prepared and WB experiment was carried out. Upper sample, electrophoresis, membrane transfer, incubation of primary antibody, anti-Histag 1:10, 000,4 ℃ overnight
2019.7.27 Sat.
No experiments, participate in the HUST exchange
2019.7.28 Sun
rest
2019.7.29 Mon
SDS-PAGE adhesive was prepared and WB experiment was carried out. Upper sample, electrophoresis, membrane transfer, sealing, incubation of primary antibody, anti-Histag 1:10,000,
2019.7.30 Tue
Wash, incubate the secondary antibody, wash, expose, X-ray film, analyze the results and find that the signal is too strong, WB needs to be done again.
2019.7.31 Wed
Prepare for meetup in hangzhou. Experiment suspended
2019.8.1 Thu - 8.4. Sun
In HANGZHOU MEETUP
2019.8.5 Mon
SDS-PAGE adhesive was prepared and WB experiment was carried out.Upper sample, electrophoresis, membrane transfer, sealing, incubation of primary antibody, optimization of reaction conditions, anti-histag 1:100,000
2019.8.6 Tue
Wash, incubate the secondary antibody, wash, expose and photograph, and obtain satisfactory experimental results
2019.8.7 Wed
Plasmid containing GFP,( plate1,4K), and 10ulddH20 were found from iGEM distribution kit, and the GFP fragment was amplified as the template. Electrophoresis verification showed that the electrophoresis result was not ideal
2019.8.8 Thu
According to the redesign of primers, repcr, electrophoresis, gel cutting and purification
2019.8.9 Fri
IGEM distribution kit linearized pSB1C3 plasmid, enzyme digestion, E/P, electrophoresis validation, gel purification
J33201 fragment was cut and purified, E/S was cut and purified
The fragment of RBS-GFP was cut and purified, enzyme digestion, X/P, electrophoresis verification, and cut and purified
T4L ligase connected the above fragments at 16℃ for 1h
The junction product transformed E. coli DH5 a, coated with a plate containing chloramphenicol resistance, and cultured overnight
2019.8.10 Sat
Positive clones grew on the plate, and the plate was put in a 4℃ refrigerator for experiment on Monday
2019.8.11 Sun
Rest and do no experiments
2019.8.12 Mon
The recombinant j33201-rbs-gfp was verified by colony PCR on the plate, PCR products were electrophoresis, positive clones were selected, chloramphenicin-resistant LB broth was inoculated, and overnight culture was conducted
2019.8.13 Tue
The bacteria solution was reserved with glycerin bacteria, the remaining small plasmid was identified by enzyme digestion, and three plasmids were selected and sent to the company for sequencing
2019.8.14 Wed
Sequencing results were in line with expectations and functional measurements were initiated. Prepare arsenic preparation, prepare mother liquor
2019.8.15. Thu
Growth curve experiment
Induction curve experiment
2018.8.16 Fri
Analysis of the data