Methylation Reaction using dam Methylase:

Reagents (NEB)

10x Methylation buffer (MB) = 50 mM Tris-HCl pH 7.5@25°C
10 mM EDTA
5 mM BME
400x S-adenosylmethionine (32 mM stock) diluted 1:1 with 1x Methylation buffer dam Methylase 8,000 U/ml

Reaction: 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.

Library with BclI linkers

BclI is from NEB @10,000 U/ml. Optimal temp is 50°C.

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.

Linker ligation: 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).

Preparation of High Efficiency Electrocompetent D1210 Cells.

Preparation of cells
  1. Inoculate 500 ml of 2XYT media (containing Streptomycin 30 µg/ml) with 1/100th volume of fresh overnight culture.
  2. Grow cells at 37°C with vigorous shaking to an ABS600 of 0.5 to 0.7. equal to about 3-4 x108 cfu/ml
  3. To harvest, chill the flask on ice for 15 minutes and centrifuge in a cold rotor at 4,000 x g max for 15 minutes.
  4. Remove as much of the supernatant as possible. Resuspend pellets in a total of 500 ml of ice-cold sterile ddH20. Centrifuge as in step 3.
  5. Resuspend cells in a total of 250 ml of ice-cold sterile ddH20. Centrifuge as in step 3.
  6. Resuspend cells in 20 ml of ice-cold sterile 10% glycerol. Centrifuge as in step 3.
  7. Resuspend cells in 1-2 ml in ice-cold sterile 10% glycerol. The cell concentration should be about 1-3 x 1010 cells/ml.
  8. Freeze the suspended cells in 50 ul aliquots on dry ice - ethanol and store at -80°C. The cells are good for at least 6 months under these conditions.
Keep the cells as close to 0°C as possible throughout their preparation

Preparation of pSCANS plasmid with or without insert.

E. coli D1210 carries a LacIq allele on the chromosome which ensures tight repression of the P1 replication origin in the pSCANS vector. Most LacIq strains carry this allele on incompatible F' plasmids, therefore they cannot be used as hosts for the F-based pSCANS vectors. D1210 is a derivative of the highly transformable strain HB101 [F- lac (i+o+z+y-) Gal- Pro- Leu- Thi- EndoI- hsm hsr recA rpsl] that also carries the lacy+ allele on the chromosome, which facilitates IPTG uptake leading to excellent, reproducible amplification of the pSCANS vector (with or without inserts) after addition of IPTG to the medium.

Template preparation.

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.