Experiments and principles
By electroshock method or caCl2, RbCl (KCl) and other chemical reagents, the permeability of the cell membrane is temporarily changed, becoming competent cells that allow foreign DNA molecules to enter. The transformation efficiency of competent cells prepared by RbCl method is high.
Experimental reagents· equipment
reagent
MnCl2
HEPES
CaCl2
SOB medium
SOC liquid medium
liquid nitrogen
equipment
Water bath pot
Triangle bottle glass bottle
Refrigerated centrifuge
Membrane
Centrifuge tubes
Weighing balances
How-to
1. LB medium preparation (solid medium plus 2% agar powder)
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Yeast powder YesastExtract
5g
Peptone Peptone/g
10g
NaCl
water
1000ml
Second, TFBI solution preparation
RbCl (Rubidium Chloride)
1M KAc (potassium acetate)
12.3ml
1M CaCl2 (Calcium Chloride)
4.1ml
1M MnCl2 (manganese chloride, pink)
20.5ml
Glycerol (glycerol)
61.5g
0.1M HAc (acetic acid)
Adjust the pH to 5.8 ml in 8 ml
Ultrapure water
Topped up to 410ml
Sterilize with 0.22 μm membrane filtration after preparation is complete
Third, TFBII solution preparation
1M MOPS (pH 6.5, solution yellow)
1.5ml
11.25ml
1M RbCl
22.5g
1M KOH (Potassium Hydroxide)
Adjust the pH to 6.5
Topped up to 150ml
Fourth, efficient competent cell preparation
1. Take dh5α strains stored at laboratory-80 °C and scribble them on antibiotic-free LB plates, incubate at 37 °C overnight for activation;
2. Pick a single colony from the LB plate, and try to select colonies with fuller colonies, smooth edges, larger heads and better growth;
Select the above single colonies and inoculate them in a 25 ml Erlenmeyer flask filled with 10 ml of antibiotic-free LB liquid medium;
4. Shake culture at 37 °C overnight (shaker speed 200 rpm, about 12h), fully activated;
5. Inoculate the above bacterial solution into a large Erlenmeyer flask in the ratio of 1:100, and incubate at 37 °C with shaking for 2 to 2.5 h (try not to time out), to OD590 to reach 0.4 to 0.6;
6. Transfer the cultured bacterial solution into a clean and sterile 50 ml centrifuge tube and place it on ice for 10 min (at this time, the configured TBFI solution ice bath can be pre-cooled, the centrifuge is pre-cooled to 4 °C, if the centrifuge cools down slowly, it is best to advance a little more);
Centrifuge at 4 °C at 4°C for 10 min and discard the supernatant;
Add 1/2.5 of the initial bacterial medium to the pre-chilled TBFI solution on ice (60 ml here if 60 ml is used earlier, here it is 60/2.5 = 24 ml), gently suspend the cells, and leave on ice for 10 min;
Centrifuge at 4 °C at 4°C for 10 min at 4000 rpm and discard the supernatant;
Add 1/25 volume of the initial bacterial medium to the pre-chilled TBFII (if 60 ml is used earlier, here it is 60/25 = 2.4 ml, that is, 1/10 of TBFI, of course, the specific volume can also be changed up and down according to personal experience), gently suspend the bacterial sedimentation;
11. Aliquot into EP tubes, each tube 100 μl (EP tubes are best pre-cooled, the dispensing process requires ice).
12. -80 °C refrigerator long-term storage, regular activation, conducive to long-term preservation. (Do not lose power during storage, otherwise the cells may be inactivated).
[ Special Notice ]
After 6-8 weeks, the conversion rate of competent cells stored in the -80 °C refrigerator is rapidly reduced, and the transformation capacity of competent cells can be identified by closed-loop plasmids of known standards and concentrations.