Gene cloning: efficient receptor cell production

Summary

Highly efficient receptor cells can be used for plasmid transformation.DNA cloning refers to the process of molecular manipulation in which a DNA fragment containing a target gene or other meaningful DNA is ligated to a self-replicating vector DNA in vitro and then transferred into a host cell or a recipient organism for expression or further research, so DNA cloning is also known as molecular cloning, gene manipulation or recombinant DNA technology.DNA cloning involves a series of molecular biology techniques, such as obtaining the target DNA fragment, selecting the vector, introducing into host cells, and screening the recombinants. DNA cloning involves a series of molecular biology techniques, such as the acquisition of target DNA fragments, the selection of vectors, the selection of various tool enzymes, in vitro recombination, introduction into the host cell and recombinant screening techniques.

Operation method

Highly efficient receptor cell production

Principle

The main principle is that the permeability of the cell membrane is temporarily changed by the electric shock method or treatment with chemical reagents such as CaCl2 and RbCl (KCl), so that the cell becomes a receptor cell that can allow the entry of exogenous DNA molecules. Since the transformation efficiency of the receptor cells prepared by the RbCl method is high, this experimental operation is illustrated as an example.

Materials and Instruments

Cell samples
MnCl2 HEPES CaCl2 SOB Culture medium SOC liquid medium Liquid nitrogen
Water baths Triangular flasks Glass jars Cryo-centrifuges Filter membranes Centrifuge tubes Weighing balances

Move

I. LB medium preparation (solid medium plus 2% agar powder) Yeast

Extract 5g

Peptone

Peptone/g 10g

NaCl 10g

water 1000ml

Second, TFBI solution preparation

RbCl (Rubidium Chloride) 5

g1 M KAc (Potassium Acetate) 12.3

ml1 M _CaCl2 (Calcium Chloride) 4.1

ml1 M _MnCl2 (Manganese Chloride, Pink) 20.5

mlglycerol (Glycerol) 61.

5g0.1 M HAc (Acetic Acid) 8 ml Adjust the pH to 5.8

ultrapure water Make up to 410 ml

Note: Prepar

ation

Note: After the preparation, filter the solution with 0.22μm membrane to remove bacteria.

TFBII solution

preparation

1 M MOPS (pH 6.5, solution is yellow) 1.5

ml1 M _CaCl2 (calcium chloride) 11.25

ml1 M RbCl 1.5

mlglycerol (glycerol) 22.5

g1 M KOH (potassium hydroxide) Adjust the pH to 6.5

ultra-pure water Make up to 150 ml

Note: After preparation, use a 0.22 μm membrane

to filter out bacteria.

Remove bacteria.

Preparation of high-efficiency receptor cells

1. Take the DH5α strain stored at -80℃ in the laboratory and line on a non-resistant LB plate, incubate at 37℃ overnight for activation.

2.

2. Pick single colonies from the LB plate, try to select colonies with fuller, smooth edges, larger heads and better growth;

3. Pick the above single colonies and inoculate them into 25 ml conical flasks containing 10 ml of non-resistant LB liquid medium;

4. Cultivate them at 37 degrees Celsius with shaking overnight (shaking machine speed of 200 rpm for about 12h) for full activation;

5. Inoculate the above bacterial solution into larger flasks at a ratio of 1: 100; 6. Inoculate the above bacterial solution into larger flasks at a ratio of 1:

100 for activation. Inoculate the above bacterial solution in the

ratio of 1: 100 to a large conical flask, 37 ℃ shaking culture 2 ~ 3h to OD590 up to 0.4 ~ 0.6;

6. will be cultured into a clean and sterile bacterial solution into a 50mL centrifuge tube, placed on ice for 10min (at this time, you can configure a good pre-cooling of TBFI solution ice bath, centrifuges should start pre-cooling, if your centrifuges cool down slower, it is best to advance a little bit more

); 7. If your centrifuge is slow to cool down, it is better to advance a little more

);

7. centrifuge at 4000rpm for 10min at 4℃, discard the supernatant;

8. add 1/2.5 of the initial bacterial medium to the pre-cooled TBFI solution on ice (if you used 60ml earlier, here it is 60/2.5=24ml), gently suspend the cells, and leave it on the ice for 10min;

9. centrifuge at 4℃, 4000rpm for 10min, discard the supernatant;

10. Add 1/25 volume of pre-cooled TBFII of the initial bacterial medium (if the previous use of 60ml, here is 60/25 = 2.4ml, that is, 1/10 of TBFI, of course, the specific volume can also be changed according to personal experience up and down), gently suspended bacterial precipitation;

11. Dispense into EP tubes, each tube is dispensed with 100ul (EP tubes are best to be pre-cooled, the dispensing process

); 12. (EP tubes should be pre-cooled, and the dispensing process

should be carried out on ice);

12. Store at -80℃ for a long time (at least one year).

Caveat

1. It is crucial to select low-generation strains, and never save the activated strains after each activation, not to mention keeping the strains at -20 degrees for a long time.

2. E. coli can be stored in the refrigerator for about one month after overnight growth on the plate; inoculation of fresh colonies or bacterial fluids is conducive to synchronous and rapid growth of bacterial cells, so that the bacterial population is in the sensory state after reaching a certain OD value (0.375) for most of the cells.

3. The growth of E. coli requires oxygen, and the purpose of vigorous oscillation is to increase the dissolved oxygen of the medium to facilitate bacterial growth.

4. It takes about 2.0 - 2.5 hours to reach the early and middle stage of bacterial logarithmic growth, this step is critical, if it exceeds this stage, the transformation efficiency will drop dramatically.

5. The purpose of low temperature operation is to make the cells stop growing and maintain their sensory state.

6. Cells are fragile when washing, so be gentle when suspending and settling, use a pipette gun to gently pipette the cells.

7. Receptor cells stored in the refrigerator at -80°C have a rapidly decreasing transformation rate after 6-8 weeks. A closed-loop plasmid such as pUC18/19 with a known standard and concentration can be used to identify the transforming ability of receptor cells.

Common Problems

In order to improve the transformation efficiency, the following factors should be considered in the experiment:

1. Cell growth status and density: Do not use cultures that have been transferred several times or stored at 4°C. The cell growth density is better when it just enters the logarithmic growth phase, which can be controlled by detecting the OD600 of the culture solution.

2. Quality and concentration of plasmid: the plasmid DNA used for transformation should be mainly superhelical DNA. transformation efficiency is proportional to the concentration of exogenous DNA within a certain range, but when too much exogenous DNA is added or the volume is too large, the transformation efficiency will be reduced. In general, the volume of DNA solution should not exceed 5% of the volume of the receptor cell.

3. Quality of reagents: The reagents used need to be of the highest purity (GR or AR) and prepared with ultrapure water, preferably stored in separate packages in a dry, cold and dark place.

4. Prevent the contamination of heterozygous bacteria and hetero-DNA: the whole process should be carried out under aseptic conditions, the utensils used, such as centrifuge tubes, pipette tips, etc., preferably new and autoclaved, all the reagents should be filtered to remove bacteria, and pay attention to prevent the contamination by other reagents, DNA enzymes, or hetero-DNA, or else it will affect the transformation efficiency or the transfer of hetero-DNA, which may cause unnecessary troubles for the subsequent screening and identification. Otherwise, it will affect the transformation efficiency or the transfer of hetero-DNA, which will bring unnecessary trouble to the screening and identification in the future.

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