Team:CIEI-BJ/Protocol
1. Prepare reaction mix according to the following table
| Enzyme digestion system (20μL) | |
| Fragment or plasmid needs to be digested | 16μL |
| Corresponding enzymes of upstream restriction enzyme cutting site | 1μL |
| Corresponding enzymes of downstream restriction enzyme cutting site | 1μL |
| Enzyme buffer (Buffer) | 2μL |
2. Incubate at recommended temperature (37℃) for at least 1 hour.
3. Purify the digestion product by inactivating at 80 ° C.
1. Prepare reaction mix according to the following table
| Fragment of insert (50ng) | 2μL |
| Liner vectors (50ng) | 2μL |
| CE Enzyme | 1μL |
| Enzyme buffer (Buffer) | 2μL |
| H2O | 3μL |
2. Incubate at recommended temperature (37℃) for at least 30 minutes.
3. Keep at 4 ° C for 5 minutes ready for transformation.
Follow the clontech protocol
Colonies on the SD-Leu/-Trp plate were selected to be resuspended in 100ul 0.9% NaCl and inoculated equally into the medium with or without AFB1 respectively. OD600 was measured every two hours.
Take some of the yeast cells respectively from the cells with or without AFB1, put them on the slide, set the exposure time at 200 ms, and take the fluorescence images under excitation luminescence.
Take 50 ml yeast liquid of different concentrations which was induced by different AFB1 respectively. Centrifuge and collect it, add same volume of glass drops and 300 uL of “cracking” buffer (PSB PH7.4, 10 %SDS，10mmNaCl), mediate and shake for 30 sec, recycle for 8 times, centrifuge at high speed andextract supernatant liquid, add appropriate amount of 5xSDS loading buffer, introduce the sample after denatured at high temperature of 99°C , run SDSpage, use wet electric membranous transformer to transform denatured protein to the pvdf membrane. Use dried skimmed milk to close it, use EYFP antibody (1:1000) for incubation at room temperature for 4 hours, then use HRP labeled secondary antibody (1:5000) and incubate for 2 hours also at room temperature, add chromogenic substrate, and expose it in the imager.
Colony PCR is a very easy method to screen clones. Resuspend a single colony in 50-100 uL LB containing antibiotic, and keep it at 37℃ and 200rmp for 2-3 hours which is used for PCR template. Prepare the following PCR mixture:
Per Reaction
| dH2O | 7 ul |
| 10uM forward primer | 1 ul |
| 10uM reverse primer | 1 ul |
| PCR template | 1 ul |
| 2xTaq Mix | 10ul |
| Total | 20 ul |
PCR condition:
1. 94℃ 4 minutes
2. 20 cycles of: 95℃ 20 seconds, 58℃ 15 seconds, 72℃ 2 minutes
4. 4℃ hold
TB solution: 10 mM Pipes, 55 mM MnCl2, 15 mM CaCl2, 250 mM KCl. (PIPES 3.021g/l, CaCl2.2H2O 2.205 g/l, KCl 18.637 g/l, MnCl2.4H2O 10.885 g/l). All ingredients except MnCl2 are dissolved and adjusted to pH 6.7 with KOH, then dissolved with MnCl2, and stored at 4℃ after filtration.
1.Inoculate E.coli to LB liquid medium, incubate at 37 ℃, 200 rpm overnight.
2.30uL E.coli liquid culture incubated overnight to 3mL LB liquid media, incubate at 37 ℃, 200 rpm for 2 h, then thaw on ice;
3.All liquid culture was centrifuged at 12,000 for 1 min. Collect the pellet and discard the supernatant;
4.Add 1 mL cold TB solution, thaw on ice for 15 min;
5.Centrifuge at 12,000 for 1 min, discard the supernatant;
6.Resuspend cells with 100uL TB solution, add 7uL DMSO, mix and transfer to ice for 10 min or store at -70℃.
1.Add 1~2 μL vector or 10uL ligation product to 100uL competent cells, then incubate on ice for 30 min;
2.Heat shock at 42℃ for 90 seconds;
3.Incubate on ice for another 2 min;
4.Add 800 μL LB liquid media, incubate at 37 ℃, 200rpm for 30 min;
5.Plate moderated transformed cells to LB agar , incubate at 37℃ for 12~16 h.