10x Methylation buffer (MB) = 50 mM Tris-HCl pH 7.5@25°C400x S-adenosylmethionine (32 mM stock) diluted 1:1 with 1x Methylation buffer dam Methylase 8,000 U/ml
10 mM EDTA
5 mM BME
50 µl vol. final vol.
DNA + TE buffer to 43.75 µl
5.0 µl 10X MB
0.25 µl 200x SAM
1.0 µl Methylase = 8U which should be sufficient to methylate 8 µg DNA in 1 hr.
Incubate overnight at 37°C, then add
0.25 µl 200x SAM
0.6 µl Methylase, incubate for 1 additional hr at 37°C.
Phenol extract and ETOH ppt.
DNA: must be dam methylated.
Fragments can be generated by CviJ1 digestion (gives blunt ends) or sheared, filled-in and phosphorylated. CviJI normally cleaves RGCY sites between the G and C leaving readily cloneable blunt ends. However, in the presence of 1 mM ATP and 20% dimethyl sulfoxide the specificity of cleavage is relaxed and CviJI also cleaves RGCN and YGCY sites. Under these "star" conditions, CviJI cleavage generates quasi-random digests which appear to yield unbiased clone libraries. Digested or sheared DNA should be size selected at this point.
VECTOR: pSCANS with stuffer fragment. Cut with BamH1and and then phosphatase at 37°C. Bam digestion releases a stuffer fragment >10 kb (see fax for gel photo) and the 4.4 kb linearized vector band. Purify vector DNA using your favorite gel clean up method. Vector should be tested by electroporation into D1210 cells to make sure the background is nil.
25 µl sized DNA
10 µl BclI oligo (phosphorylated, 500 µM = pGCGTGATCACGC)
10 µl 5X ligation buffer (with PEG and ATP same as Gibco's)
5 µl ddH20.
heat at 68° for 30 seconds, chill, spin
add 2 µl T4 DNA ligase, incubate at 14°C overnight.
Heat inactivate ligase 15 min at 68°C. Add 2.5µl BclI, 50°C for 5 hrs.
Phenol chloroform extracte, ETOH ppt. using 1/3rd volume of 7.5M ammonium acetate and 2.5 volumes of 100% ethanol. Spin, wash with 70% ETOH, dry, rehydrate in 50 µl ddH20.
To size select and eliminate excess linkers at this point run entire sample on a low melt agarose prep gel and cut off the bottom of the gel (e.g. below 2 Kb). Put gel in a clean rig, add new buffer and reverse polarity until band is reconcentrated.
Cut out band and melt in 1X NEB#3, incubate with 1µl BclI at 50°C for 1 hr. to digest any residual BclI linker. Purify DNA using your favorite gel clean up method. We use GFX spin columns (Amersham Pharmacia) which also help to remove any residual traces of the BclI linker.
Ligate vector and BclI linkered DNA fragments overnight at 14°C. Typically we do a 25 µl ligation reaction. Dilute to 50 µl with 1X NEB#3, heat inactivate ligase 20 min at 65°C, cool add 1 µl BclI incubate at 50°C for one hr to cleave any chimeric clones (i.e. ones with more than one insert). Add 1 µl Plasmid-Safe-ATP dependent DNase (Epicentre Technologies 10 U/µl) and continue to incubate for an additional 1 hr at 50°C to digest away any BclI cut molecules or residual linearized pSCANS vector.
Phenol chloroform extract, ETOH ppt. wash with 70% ETOH, dry, resuspend in 10 µl ddH20.
Electroporate 5 µl into 50 µl D1210 cells. Plate on prewarmed 2XYT Kan (50 µg/ml) Strep (30 µg/ml) plates. Incubate at 37°C overnight.
Alternatively, pSCANS (T/A) with double XcmI sites is cut with XcmI leaving single 3' dT overhangs at each end of the linearized vector. Ligate with sized DNA fragments that have had single dA residues added to their 3' ends by incubation with dATP and Taq DNA polymerase. (see Kwak JH, Kim MY Construction of T-vector for direct cloning and expression of cloned genes in Escherichia coli. Anal Biochem 228:178-80 (1995) and Kawata Y, Yano S, Kojima H Construction of a genomic DNA library by TA cloning Biotechniques 24:564-5 (1998).
Bacterial colonies containing library plasmid are plated on 2XYT plus kanamycin plates. Colonies are picked and grown in 1 ml of 2XYT with shaking at 37° C without addition of IPTG for 5 hrs in 96 deep-well plates. An aliquot of each culture well is then mixed with an equal vol. of 20% glycerol and the samples are archived at -70° C in 96-well microtiter dishes in case a clone needs to be regrown at some future date. IPTG (1 mM final conc.) is added to the remaining portion of each culture well to induce plasmid amplification and incubation is continued overnight. High quality sequencing templates are prepared using an alkaline lysis protocol, followed by neutralization in the presence of a proprietary ion exchange resin (Edge BioSystems, Gaithersburg, MD) to bind cellular debris, which are then removed by filtration, and plasmid DNA is recovered by isopropanol precipitation. All steps in the procedure are performed in a 96-well format as are the resulting sequencing reactions.