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[Principle of experiment]
Competence means that the bacteria are in a state where they easily absorb foreign DNA. Transformation refers to the process in which plasmid DNA or recombinants constructed with it as a vector lead human bacteria. The principle is that the bacteria are in a low-osmotic CaCl2 solution at 0 ° C, and the bacterial cells expand into a spherical shape. The DNA in the conversion mixture forms a DNase-resistant hydroxy-calcium phosphate complex which adheres to the cell surface and undergoes a short-time heat shock treatment at 42 ° C to promote the cell to absorb the DNA complex. Place the bacteria in a non-selective medium for a period of time to promote the expression of new phenotypes (such as Ampr, etc.) obtained during the transformation process, and then apply this bacterial culture to the selective medium containing ampicillin .
After transforming the recombinant plasmid into the host cell, the transformed colonies need to be screened and identified. Screening using alpha complementation is the most commonly used identification method. Many vectors in use today have a promoter of E. coli β-galactosidase and a DNA sequence encoding an α-peptide chain. This structure is called lacZ gene. The alpha peptide chain encoded by the lacZ gene is a short fragment (146 amino acids) at the amino terminus of beta galactosidase. When any plasmid vector carrying the lacZ gene is transformed into an E. coli cell with such a β-galactosidase mutation in the chromosomal genome, a functionally active galactosidase will be produced in IPTG (isopropyl β-D —Thiogalactoside), blue colonies (galactosidase can form) are formed on the medium plate containing Xgal (5-bromo-4-chloro-3-indole-6-D-galactoside) The colorless compound Xgal was cut into galactose and the dark blue substrate 5-bromo-4-indigo). When a foreign DNA fragment is inserted into the polyclonal site in lacZ, it will destroy the reading frame of the α-peptide chain, so that it cannot synthesize an active α-peptide complementary to the mutant β-galactosidase in the recipient bacteria. , Resulting in the inability to form functionally active β-galactosidase, it can not decompose Xgal and appear blue, so clones containing recombinant plasmid vectors are often white colonies.
[Instruments, Materials and Reagents]
(1) Instruments and materials
Ultra-clean workbench, cryogenic centrifuge, thermostatic shaker, thermostat, thermostatic water bath, centrifuge tube, test tube, petri dish, conical flask, inoculation needle, glass coated stick, alcohol lamp, tweezers, toothpick, E. coli DH5a, plasmid
(2) Reagents
0.1mol / L CaCl2 solution LB liquid medium LB solid medium
Ampicillin (Amp): Prepare 100mg / mL solution with sterile water and store in -20 ℃ refrigerator.
Xgal: Dissolve Xgal in dimethylformamide and make up 20mg / mL. No need to filter and sterilize. Pack in small packages and store at -20 ℃ in dark place.
IPTG: Take 2g of IPTG dissolved in 8mL of double-distilled water, then make up to 10mL with double-distilled water, filter and sterilize with 0.22um filter membrane, 1mL each, store at -20 ℃.
[Experimental steps]
(1) Preparation of competent cells
1. Aspirate 15µl of E.coil bacterial liquid, inoculate in 20ml LB liquid medium, and culture at 37 ° C with shaking overnight. After OD600 = 0.5, transfer the bacterial suspension to 50ml LB liquid medium at an inoculation amount of 1:50. Shake at 37 ° C to expand the culture. When the culture solution begins to appear turbid, measure OD600 every 20-30min to OD600 = 0.6, and stop the culture.
2. Transfer the culture solution into a centrifuge tube and centrifuge at 5000 rpm for 10 minutes at 4 ° C in an ice bath for 10 minutes.
3. Discard the supernatant and gently suspend the cells with 4ml of ice-cold 0.1M CaCl2 solution until uniform, and place on ice for 30min.
4. Centrifuge at 5000 rpm for 6 min at 4 ° C.
5. Discard the supernatant and gently suspend the cells with 2ml of ice-cold 0.1M CaCl2 solution to uniformity. After placing on ice for a while, a competent cell suspension is prepared.
6. The above prepared competent cell suspension can be placed on ice and used directly in transformation experiments within 24 hours, or 15% autoclaved glycerin can be added, mixed, and packed in 1.5ml centrifuge tubes , 100µl of competent cell suspension per tube, stored at -70 ℃. .
(2) Transformation of plasmid DNA into E. coli
1. Take 100µl of the competent cell suspension after shaking (if it is a cryopreservation solution, you need to perform the following operation immediately after thawing), add 5µl of the conjugated product, gently shake it, and place it on ice for 30min, in a 42 IPTG water bath Insulate for 90s, then quickly cool on ice for 2min.
2. Add 900µl of LB liquid medium, the total volume is about 1ml, mix and shake at 37 ° C for 90 minutes to restore the normal growth of the recipient bacteria and make the transformants resistant to Ampr.
3. Add 40 uL of 20 mg / mL Xgal and 4 uL of 200 mg / mL IPTG solution to the pre-made LB agar plate, and use a sterilized glass fader (alcohol lamp burned and cooled) to evenly coat the agar gel surface Absorb at 37 ° C.
4. Centrifuge the recovered cells at 4000 rpm for 5 min, remove the upper layer of 900 µl LB medium, and resuspend the bacterial cells with the remaining 100 µl until uniform.
(4) Inspection of α complementary phenomenon
The resuspended cells were evenly spread on the LB plate containing X-gal + IPTG + ampicillin, cultured upside down at 37 ℃ for 12-24h, and colonies appeared. The white colonies are theoretically recombinant clones.
For further verification, multiple white colonies can be picked and inoculated into 1 ml of LB liquid medium containing ampicillin, cultured at 37 ° C for 6-8h with shaking, and then extracted with plasmids and verified by enzyme digestion or colony PCR amplification.
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