反应详情

catarrhalis cell pellet was resuspended in 20 mL Tris-EDTA (TE) buffer, pH 7.5. Pronase (final concentration 500 μg/mL) and SDS (final concentration 1%) were added and the suspension was incubated in a 37° C. water bath for 2 hours. DNA was isolated by sequential extractions with phenol (1 time), phenol-chloroform (1:1, 2 times), and chloroform-isoamyl alcohol (24:1, 1 time). Extracted DNA was dialyzed against 1M NaCl at 4° C. for 4 hours. This was followed by dialysis against TE buffer, pH 7.5, at 4° C. for 48 hours (3 buffer changes). DNA was ethanol precipitated from the dialysate. Large size DNA was collected by spooling on a glass rod. Air dried DNA was dissolved in 3 mL water. Small scale Sau3A (New England BioLabs) restriction digests of chromosomal DNA (final volume 10 μl) were done to establish conditions required to obtain maximal amounts of chromosomal DNA with a size range of 15-23 kb. Large scale digests were prepared once the optimal digestion conditions were defined. The large scale digests consisted of 50 μl chromosomal DNA (290 μg/mL), 33 μL water, 10 μL Sau3A buffer (New England BioLabs), 1 μL BSA (10 mg/ml, New England BioLabs) and 6.3 μL Sau3A (0.04 U/μL), and were incubated at 37° C. for 15 minutes. Reactions were stopped with the addition of 10 μL 10X loading buffer (100 mM Tris-HCl pH 8, 10 mM EDTA, 0.1% bromophenol blue, 50% glycerol). Digested DNA was applied to 0.5% agarose gels (prepared in Tris-acetate-EDTA (TAE)) and separated according to size at 50 V for 6 hours. The region of the gel encompassing DNA of size 15-23 kb was cut from the gel and placed in dialysis tubing (BRL) with 3 mL TAE. DNA was electroeluted from the gel slice overnight at a field strength of 1 V/cm. Electroeluted DNA in TAE was extracted once with phenol, once with phenol-chloroform (1:1), and finally precipitated with ethanol. The dried DNA pellet was dissolved in 5 μL water. Size-fractionated chromosomal DNA was ligated with BamHI cut EMBL3 arms (Promega) using T4 DNA ligase in a final volume of 9 μL. The entire ligation reaction was packaged into phage λ using a commercially purchased packaging kit following the manufacturer's (Amersham) protocol.

1. customwas incubated in a 37° C.
2. customfor 2 hours