Team:NJTech China/Notebook
Agarose Gel Electrophoresis
1% agarose is added to 1 × TBE and heated in microwave for about 1 min to fully dissolve the agarose. Let it cool for a while, add a small amount of ExGreen nucleic acid dyeμL, mix them thoroughly and pour the contents into the rubber plate. The gel is verified and a single band appeared, indicating that the plasmid is linearized.4μL per well, 1μL 10× Loading Buffer per well.
Electrophoresis : 125V 30min
BCA protein assay
Beyotime BCA Protein Assay Kit
Cell Lysis
1) Divide the bacterial solution into 50mL centrifuge tubes. Centrifuge at 6000 rpm at 20°C for 10 minutes and pour the supernatant into a waste bucket.
2) Resuspend the cell pellet in 10 mL 10 mM Tris-HCl (pH 7.5), centrifuge at 6000 rpm at 20°C for 10 minutes , and pour out the supernatant. Repeat the above steps again.
3) After centrifugation, the cell precipitation was resuspended in 9.9 mL 10 mM Tris-Hcl (pH 7.5) and 0.1 mL lysozyme , and incubated at 30℃ for 20 minutes.
4) 200 μl 100 mM PMSF was added to the fermentation broth. Remove 200 μl liquid into 2ml centrifuge tube and mark it.
5) Immerse the bacterial suspension in ice and perform sonic treatment with 3 s sonic wave and 3 s interval for a total of 30 minutes.
6) Remove 200 μl of whole protein solution from 50mL centrifuge tube into 1.5mL centrifuge tube and mark it.
7) Centrifuge at 4℃ and 6000 RPM for 20 minutes, take 200 μl supernatant from the 50mL centrifuge tube into the 2 mL centrifuge tube and mark it. Pour the remaining supernatant into the new 10 mL centrifuge tube and wait for ultrafiltration. The precipitate was resuspended with 300 μl pure water in a 2mL centrifuge tube and marked.
IPTG Protein Expression Induction
1) Transform expression plasmid into BL21. Plate on antibiotic selection plates and incubate overnight at 37°C.
2) Resuspend a single colony in liquid culture with antibiotic to produce a starter culture. Inoculate starter culture into 50 ml expression media containing antibiotic.
3) Incubate at 37°C with shaking at 220 rpm until OD600 reaches 0.6 .
4) For most vector systems, induce with 250 μM IPTG and express protein for 24 hours at 16°C with shaking at 220 rpm.
PCR
50μL system (2 × Rapid Taq Master Mix):
2 × Rapid Taq Master Mix 25.0 μl
Primer1 (10 μM) 2.0 μl
Primer2 (10 μM) 2.0 μl
Template DNA* x μl
Add ddH2O to 20μL
*For a 50 μL reaction system, the recommended input amount of template is as follows:
| Template type | Template start amount |
| Genomic DNA of animals and plants | 0.1-1 μg |
| Genomic DNA of E. coli | 10-100 ng |
| cDNA | 1-5 μl (< 1/10 of the reaction volume) |
| Plasmid DN | 0.1-10 ng |
| λDNA | 0.5-10 ng |
Program:
| Loop step | Temperature | Time | Number of cycles |
| Pre-denatured | 95℃ | 3min | 30-35cycles |
| Changing nature | 95℃ | 30sec | |
| Annealing | 53℃ | 30sec | |
| extend | 72℃ | 15sec | |
| Extend comletely | 72℃ | 5min |
Product Collection And Purification
Ultrafiltration：
1) Take appropriate amount of fermentation solution, disperse them into two 50mL centrifuge tubes, centrifuge at 3500 rpm, 10°C for 10 min after equilibration.
2) After centrifugation, the supernatant is poured out, and 50 μL of the supernatant is taken and marked as sample 1;
Take a certain amount of ultrapure water, resuspend the bacteria , and marked it as sample 2;
The remaining supernatant is subjected to ultrafiltration (the membrane is placed in a 35 mL centrifuge tube):
(1)Add 10 mL of ultrapure water to each of the two ultrafiltration tubes, balance, 3500 rpm, and centrifuge for 20 min;
(2)Add 10 mL of sample 1 to each of the two ultrafiltration tubes, balance, 3500 rpm, centrifuge for 20 min, aspirate the liquid on the filter, and transfer to a 1.5 mL centrifuge tube;
Repeat the operation of step 2 until all the samples 1 are processed, the liquid in the 1.5 mL centrifuge tube is marked as sample 3, and the liquid in the 35 mL centrifuge tube (i.e. the supernatant after ultrafiltration) is marked as sample 4;
(3) Add 10 mL of ultrapure water to each of the two ultrafiltration tubes, level, 3500 rpm, centrifuge for 20 min (clean filter), and immerse the filter in ethanol.
SDS-PAGE verification
Beyotime SDS-PAGE Gel Preparation Kit ; Gels were washed in pure water and then stained with 0.25% Coomassie Brilliant Blue to reveal protein bands.
Transform BL21
1) Take the competent BL21 from the -80 °C refrigerator and thaw it on ice.
2) Add the 1-2 μL DNA fragments to be transformed into 50 μL competent cells, mix the walls of the tube (do not shake and mix), and incubate them on ice for 30 min.
3) Heat-shocking for 90 sec in a 42°C water bath, then immediately set it on ice for 2 min. Do not shake the tube.
4) Add 800 μL LB medium (without antibiotics), shaking at 37°C, 200 rpm for 1 h.
5) 6000rpm, centrifuge for 2 min, discard 800μL of supernatant, suspend the bacteria body with the remaining medium, and evenly coat on the LB solid plate containing the corresponding antibiotics。
6) Incubate the plate in a 37 °C incubator for 10 min. After the bacteria solution is completely absorbed, invert the plate and incubate it overnight.
3.Plasmid Extraction：ZOMANBIO Fast Plasmid Miniprep Kit