Team:GZHS-United/Notebook
Notebook
2019.7.20
Configure TBE, liquid LB and EDTA
2019.7.21
Gene PCR amplification
2019.7.22
1. The PCR products were subjected to agarose gel electrophoresis, gel recovery and product purification
2. Amplified Arabidopsis APX
2019.7.23
1. Fragment recovery of arabidopsis APX
2. Enzyme cutting target segments
3. Extract plasmid pet28a-rpij
2019.7.24
1. Enzyme digestion vector was tested by electrophoresis and the enzyme digestion product was purified again
2. Enzyme linkage vector and target fragment
2019.7.25
Heat shock transformation of enzyme coupling products, plate culture (no colony was grown, and the analysis should be the wrong enzyme cutting site in the primer)
2019.7.26
Re-amplification of APX(unamplified bands)
2019.7.27
Re-enzyme linked plasmid to target fragment, reheated transformation, coated plate.
2019.7.28
1. No bands were visible on electrophoresis today (colony PCR and PCR purified products were marker). After replacing the dye, streaks were found on electrophoresis. After analysis, it is considered that the SYBR Green dye packed in small tubes may have problems, and the dye should be replaced on the second day.
2019.7.29
1. Replace the nucleic acid dye and repeat yesterday's colony PCR experiment
2. Pet28a-rpij plasmid was tested
2019.7.30
1. Check whether there is any problem with the plasmid. Pet28a-rpij is correct after re-sequencing
2019.7.31
1. Reprepare 5xTBE, use imidazole and Na2HPO4, prepare loading buffer, 0.22um filter membrane, and filter PBS buffer.
2019.8.5
1. Heat shock transformation of pet28a-apx3-1 (zooxanthellae), the receptive state used was DE3, and the next day, it was small shake (no bacteria) according to the situation.
2. Colony PCR (no positive bands) was performed for T4 junction products.
2019.8.6
1. Preparation of plasmid maxiprep
2. Reheat control plasmid and APX3-1
3. The vector pet28a-apx (arabidopsis thaliana) -his was constructed
2019.8.7
1. Bacteria culture of pet28a-apx3-1 (zooxanthellae)
2. Pet28a-apx (arabidopsis thaliana) plasmid was extracted
2019.8.18
0. Sterilization of all required materials (tri-steam water, blue nozzle, large nozzle, blue cover centrifuge tube, glass tube, 2-YT)
1. Parts： shake five bacteria (1O,8P,9L,10C+pSB1C3), two tubes each, 5mL LB (Chl) each
2. There are already zooxanthellae APX, arabidopsis APX, liquid which contain cultivated bacterias, and sterilizing sealed 500mL conical flask (using four), each of which can shake 150mL 2-yt (Kana) and inoculate 1.5ml bacterial liquid
→ apx3-1 cultivate again, other samples have been induced completely → apx3-1 is inducing overnight (8.20.)
→ measure precipitation and supernatant, using a large colorimetric cup
5. Decolorization and review of PAGE glue samples, it seems that there is a certain amount of exogenous protein expression in precipitation and supernatant
2019.8.19
1. Parts plasmid extraction, the concentration is almost the same as that extracted yesterday, the reason is unclear (kit?) (pollution?)
2.2-YT preparation (ZMX)
3.LB (Chl) prepare (DZH) on plate
Heat shock transformation 9L, 10C to BL21, heat shock transformation apx3-1, apx3-2 to BL21 and DE3)
5. Heat shock transformation 10L to BL21, 8P, heat shock transformation apx3-3, arabidopsis APX to BL21 and DE3
2019.8.20
1. Take precipitation and supernatant to measure, using a large colorimetric cup to do APX enzyme activity
2. Four parts plasmids were sent to test in the morning. Only testing one reaction to exclude the possibility of miscellaneous bacteria
3. The united gold award parts primers are checked and gived to the company for synthesis
4. Cultivate the bacteria according to the growth of bacteria, or re-transform the coating plate
From August to September, we continue doing Prokaryotic protein expression and measurement of enzyme activity.