1. Pre-cool two 14-ml BD Falcon polypropylene round bottom centrifuge tubes on ice. One is used to transform the sample, one is used as the pUC18 control. The NZY+ broth medium is preheated at 42 °C.
2. Melt the competent cells on ice. Gently mix the cells and dissolve them into 100 ul/tube.
3. Add 4 ml of b-ME (mercaptoethanol) supplied with the kit to each tube.
4. Rotate the tube gently and place the cells on ice for 10 minutes, gently rotating every 2 minutes.
5. Add 0.1–50 ng of sample DNA per cell (or 2 ml of ligation reagent) (see DNA Quality and Volume Description.) Dilute control pUC18 DNA 1:10 with sterile dH2O water and take 1 ml of diluted pUC18 DNA is added to transformed cells.
6. Rotate the tube gently and place it on ice for 30 minutes.
7. The test tube is heat-shocked for 30 seconds in a 42 ° C water bath. The heat shock time is extremely important for the conversion efficiency.
8, test tube ice bath for 2 minutes.
9. Add 0.9 ml of preheated (42 °C) NZY+ broth to each tube and incubate for 1 hour at 37 ° C, 225–250 rpm shaker.
10. Take 200 ml of the transformed mixture and plate the LB agarose plate with the corresponding antibiotics (if you want to color filter, you need to add IPTG and X-gal). For the pUC18 positive control, take 5 ml of ampicillin LB agarose plate.
Note: The cells will accumulate after centrifugation at 1000 rpm for 10 minutes. The cell pellet should be resuspended in 200 *l NZY+ broth. If the cells used for plating are <100 ml, the cells are first added to 200 ml of medium and then sterilized. Coated bar plating. If using 100 ml cells, you can directly plate. Tilt the coating stick and gently tap to minimize the cell residue on the coating stick.
11. Culture at 37 ° C overnight. For color screening, please refer to the following Blue-White Color Screening operation guide.
12. The pUC18 positive control is expected to have about 250 clones (?5 × 109 cfu/mg pUC18 DNA). The conversion efficiency of the sample DNA varies greatly depending on the amount and composition of the DNA (supercoiled or ligated product), large plasmid and Non-supercoiled DNA tends to get fewer clones.
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