重组抗体制备系统

表达性宿主菌XL1-Blue

MATERIALS PROVIDED

Storage: XL1-Blue cells must be placed immediately at the bottom of a –80°C freezer directly from the low temperature shipping container. Do not store the cells in liquid nitrogen.

BACKGROUND

The XL1-Blue strain allows blue-white color screening for recombinant plasmids and is an excellent host strain for routine cloning applications using plasmid or lambda vectors.

XL1-Blue Genotype: recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´ proAB lacIqZΔM15 Tn10 (Tetr)].

(Genes listed signify mutant alleles. Genes on the F´ episome, however, are wild-type unless indicated otherwise).

XL1-Blue cells are tetracycline resistant. XL1-Blue cells are endonuclease (endA) deficient, which greatly improves the quality of miniprep DNA, and are recombination (recA) deficient, improving insert stability. The hsdR mutation prevents the cleavage of cloned DNA by the EcoK endonuclease system. The lacIqZΔM15 gene on the F´ episome allows blue-white color screening.

TRANSFORMATION PROTOCOL

1. Pre-chill two 14-ml BD Falcon polypropylene round-bottom tubes on ice. (One tube is for the experimental transformation and one tube is for the positive control.) Preheat SOC medium to 42°C.

2. Thaw the prepared competent cells on ice. When thawed, gently mix and aliquot 100 µl of cells into each of the two pre-chilled tubes.

3. Swirl the contents of the tubes gently. Incubate the cells on ice for 10 minutes, swirling gently every 2 minutes.

4. Add 0.1–50 ng of the experimental DNA to one aliquot of cells and add 1 µl of the positive control DNA to the other aliquot. Swirl the tubes gently.

5. Incubate the tubes on ice for 30 minutes.

6. Heat-pulse the tubes in 42°C water bath for 45 seconds. The duration of the heat pulse is critical for maximum efficiency.

7. Incubate the tubes on ice for 2 minutes.

8. Add 0.9 ml of preheated SOC medium and incubate the tubes at 37°C for 1 hour with shaking at 125–150 rpm.

9. Plate ≤200 µl of the transformation mixture on LB agar plates containing the ampicillin (and containing IPTG and X-gal if color screening is desired). For the positive control transformation, plate 5 μl of the transformation mixture on LB–ampicillin agar plates.

10. Incubate the plates at 37°C overnight (at least 17 hours for blue-white color screening). Colonies containing plasmids with inserts will remain white, while colonies containing plasmids without inserts will be blue. The blue color can be enhanced by incubating the plates for two hours at 4°C following the overnight incubation at 37°C.

11. For the experimental DNA, the number of colonies will vary according to the size and form of the transforming DNA, with larger and non-supercoiled DNA producing fewer colonies.