How to build a BAC library: (Ung-Jin Kim)
The most important aspect of our cloning vectors is that they are based on the E. coli F-factor replicon. It allows for strict copy number control of the clones so that they are stably maintained at 1-2 copies per cell. The stability of the cloned DNA during propagation in E. coli host is substantially higher in lower copy number vectors than in multi-copy counterparts (Kim et al, NAR, 20(5):1083-1085). The stabilizing effect of BAC and Fosmid vectors is notable especially for certain genomic DNA that are normally unstable in high copy number vectors. This includes genomes of Archaeal, mammalian, or other origins. Stable Fosmid libraries have been generated from the genomes of an Archaeum and Sea urchin that were highly unstable/unclonable in multicopy cosmid vectors. The pBeloBAC11 vector allows lacZ-based positive color selection of the BAC clones that have insert DNA in the cloning sites at the time of library construction. Because the vector exist in single copy in E. coli, purifying the DNA in large quantity takes some effort. Therefore, we have been supplying to any interested party the vector as the E. coli strain that carries the vector. Please see the experimental protocol below to find out how to prepare large amounts of pure pBeloBAC DNA. The nucleotide sequence is also available. The pBAC108L vector is the very first version of BAC vector. After transformation, clones carrying human DNA insert had to be selected by colony hybridization with labeled human DNA. Click here for its nucleotide sequence. The pFOS1 vector was a single copy cosmid vector constructed by fusing pBAC108L and pUCcos (a pUC vector in which the region including lacZ and multiple cloning sites was replaced by lambda cos sequence). In vivo homologous recombination between two vectors via cos sites resulted in pFOS1. The vector is extremely unstable in most of E. coli strains due to the presence of double cos sites. pop2136 strain (Methods in Enzymology vol.152 pp173-180, 1987), for no apparent reason, can maintain pFOS1 (and other double-cos cosmid vectors) with some stability. The bireplicon is driven by the pUC replication origin, and exists in high copies in E. coli. After in vitro packaging and transfection to E. coli, the structure of Fosmids is exactly the same as pBAC108L clones except the size; therefore Fosmids are mini-BACs with 40 kb inserts. Fosmid library can easily be constructed using the protocol for constructing cosmid libraries with double-cos vectors. The Fosmid system is useful for quickly generating miniBAC libraries from small amounts of source DNA, such as flow-sorted chromosomal DNA. Aliquots of CsCl purified pFOS1 DNA has always been made available for anyone interested in constructing Fosmid libraries. Click here for the nucleotide sequence. Inquiry should be forwarded to: Ung-Jin Kim or Hiroaki Shizuya.
PREPARATION OF BAC VECTOR DNA
Because BAC vectors are single copy plasmids, it is rather difficult to obtain large amount of BAC vector DNA. Extra care is also needed to minimize the contamination of E. coli DNA that consists more than 99% of the total DNA. However, by carefully following the procedure provided below, it is possible to obtain from a liter culture a few micrograms of pBeloBAC11 (7.5 kb), which is normally enough for library construction. 1)Starting from a single colony, grow E. coli strain containing pBeloBAC11 vector in 3 liters of LB + chloramphenicol (15 ug/ul) with good aeration overnight. Make sure to take a blue colony on an X-gal/IPTG plate. 2)Harvest the cells by centrifugation, and resuspend the cell pellet in Solution I (without lysozyme). Use 25 ml Solution I per liter culture. 3)Add lysozyme to 2.5mg/ml, and mix by inversion. 4)Add Solution II (50 ml per liter culture) and mix well by inversion. Leave on ice for 10 minutes. 5)Add 37 ml of Solution III per liter culture. Mix gently by swirling. Keep on ice for 10 minutes. 6)Centrifuge 30 minutes at 8,000g or higher at 4°C. 7)Decant the supernatant and filter it through several layers of cheesecloth. Add the RNase to a final concentration of 0.1 mg/ml, and incubate at room temperature for 15-30 minutes. 8)Using 4 Qiagen-tip 500, pre-purify the supernatant as instructed by the Qiagen procedure. Qiagen tips are pre-equilibrated with QBT, then the supernatant is applied, then washed with large volumes of QC, and eluted by 15 ml of QF per column. 9)Precipitate the DNA by adding 0.7 volume of isopropanol, mix, and centrifuge 15,000 xg for 30 minutes at 4°C. 10)Wash the DNA pellet with ice cold 70% ethanol, and air dry. 11)Resuspend DNA in 18.6 ml of TE Add 20.5g CsCl and dissolve. This is to be spun in two tubes in Beckman 70.1Ti rotor. 12)Add 0.4 ml of EtBr (10 mg/ml), mix and perform ultracentrifugation for 2-3 days at 45,000 rpm in a Beckman 70.1Ti rotor. 13)Two bands should be visible under U.V. Isolate the lower band, extract with isoamylalcohol 3-4 times, and dialyze for a few hours in TE at 4°C. 14)Ethanol precipitate DNA, rinse the pellet with 70% ethanol, and dissolve DNA pellet in TE, and store at -20°C. Solution I: 25 mM TrisHCl, pH 8.0; 50 mM Glucose Solution II: 0.2 N NaOH; 1% SDS Solution III: 5 M Potassium Acetate, pH 4.8. Add glacial aceticacid to a solution of 3 M potassium acetate to achieve a pH 4.8.
PREPARATION OF SOURCE DNA
LIGATION AND ELECTROPORATION
DNA should be in low melting agarose, in TAE or stored in 0.05M EDTA. Dialyze the sample in 50 ml tube at 4°C against 1 X TE, 1 X PA for 3-5hr with one change of solution. Melt agarose at 65°C for 10 minutes, transfer tube to 44-45°C water bath. Add agarase, using about 1.5 U for each 100 µl of melted gel. Digest 1 hour at 45°C. Set up ligation with an approximate molar ratio of vector to insert of 10:1. Every time a new batch of DNA is used it is a good idea to set up trial ligations with varying amounts of vector given the difficulties of determining the concentration of insert DNA with certainty. A typical reaction would contain 100 ng insert DNA with an average size of 200 kb and 36.5ng vector in a volume of between 120 and 150µl. Reaction Mixture: 100 µl DNA 1.8 µl pBAC (20 ng/ml) 12.0 µl 10 X ligation buffer 2.0 µl 10X PA 0.5 µl ligase 400U/ul 3.7 µl H2O Combine insert DNA, vector, PA, and H2O. Heat 5 minutes at 65°C, cool on ice. Add ligase buffer and enzyme. Mix by slowly stirring contents. Incubate overnight at 16°C. After ligation, carry out drop-dialysis of sample against approximately 25 ml 0.5 X TE, 1 X PA for 2 hours at room temperature in a 100 mm petri dish. 1 X PA is a mixture of spermine and spermidine which has a combined concentration of 1 mM (Spermidine-4HCl MW 254.6, Spermine-3HCl MW 348.6). Dissolve both in water, filter sterilize. Store frozen aliquots at -20°C. [100 X stock = Spermidine 75 mM (0.19g/10ml) + Spermine 30 mM (0.104g/10 ml); 1000 X stock = Spermidine 750 mM (1.9g/10ml) + Spermine 300 mM (1.04g/10 ml)]
PREPARATION OF COMPETENT CELLS AND BAC ELECTROPORATION 1. PREPARATION OF CELLS
1) Inoculate flasks of SOB (without Mg++) by diluting a fresh saturated (overnight) culture of DH10B 1:1000 (i.e., 0.3 ml to a flask containing 300 ml medium). 2) Grow with shaking at 37°C until OD550 reaches 0.7 (no higher than 0.8). This should take approximately 5 hr when shaken at 200 rpm. 3) Harvest cells by spinning in GSA rotor for 10 minutes at 5,000 rpm. 4) Resuspend pellet in a volume of 10% sterile glycerol equal to the original culture volume. 5) Spin 10 minutes at 5,000 rpm at 4°C. 6) Carefully pour off supernatant (pellet will be quite loose) and resuspend cells again in 10% glycerol equal to the original culture volume. 7) Spin 10 minutes at 5,000 rpm at 4°C. 8) Carefully pour off supernatant, resuspend cells in the volume of glycerol remaining in the centrifuge bottle. Pool the cells in one small centrifuge tube. 9) Spin 10 minutes at 7,000 rpm in SS34 rotor. 10) Pour off supernatant and resuspend cells in 10% glycerol, using a volume of 2.0 ml per liter of initial culture. 11) Aliquot to microfuge tubes (100-200 µl per tube) and freeze quickly in a dry ice-ethanol bath. Store cells at -70°C. 2. ELECTROPORATION
1) Wash and UV sterilize cuvettes, place on ice and prepare culture tubes with 0.5 ml SOC. 2) Thaw cells and aliquot 25-30 µl to microfuge tubes on ice. 3) Add 1-3 µl of ligation mix, and gently mix by flicking tube bottom with finger. 4) Transfer to cuvette and wipe cuvette dry. 5) Electroporate using settings of 100 Ohms, 2.5 kV, and 25 µFa. This usually gives a time constant of approximately 2.4 msec. 6) Immediately rinse contents of cuvette with SOC and transfer to culture tube using a sterile Pasteur pipet. 7) Shake for 45 minutes at 37°C. Spread on LB plates containing 12.5 µg/ml chloramphenicol, 50 µg/ml X Gal and 25 µg/ml IPTG.
Purification of BAC DNA via mini-preps
A major advantage of working with BAC clones is the ease with which pure BAC DNA can be isolated via miniprep methods. Alkaline lysis is superior to boiling methods, producing higher yields with greater reproducibility, though a significant amount of the DNA may be nicked by the alkaline treatment and coverted from supercoiled to open circular moleucles. While the low copy number of BACs means that relatively much less DNA is recovered than from multi-copy vectors, sufficient DNA can be obtained from a few ml of bacterial culture for restriction analysis, hybridization, FISH or PCR. Because the BACs are supercoiled, they are resistant to shear-induced breakage during the isolation, hence even BACs as large as 350 kb require no extraordinary measures in handling the DNA. Although we avoid vortexing the miniprepped DNA, it may be pipetted using regular pipet tips without any detectable damage to the DNA. As with large scale preparations, the smaller amount of BAC DNA relative to the amount of chromosomal DNA and protein in the cell, means that the BAC DNA will be less pure than mini-prepped DNA representing higher copy vectors. This has two consequences. First, contaminating chromosomal DNA may represent a few per cent of the yield. Second, the DNA is not particularly stable as large molecules, presumably due to nucleases present in the sample. Thus we notice degradation of the DNA after storage for only a few days both at -20 and 4°C. This degradation is apparant as an inability to generate full length molecules after restriciton digestion. Phenol extraction of the samples did not entirely prevent this degradation. More consistant recovery of BAC DNA, as well as higher yields and greater purity may be obtained using the Autogen 740 automated DNA extraction instrument (Integrated Separation Systems, Natick, MA) describe. In contrast to BAC DNA prepared manually, DNA prepared by the Autogen 740 may be analyzed after more than 10 days of storage at 4°C. Alkaline lysis mini-preps of BAC DNA We perform the following steps on up to 24 samples simultaneously. Unless stated, pauses or incubations are not needed between each step. Typical yield of BAC DNA from 3 ml cultures is 100-200 ng. 1) Inoculate a colony into a 10 ml culture containing 1.5 ml LB+ 12.5µg/ml chloramphenicol. 2) Grow overnight at 37°C by shaking at 200 rpm. 3) Transfer the culture to a 1.5ml microfuge tube. 4) Pellet the cells by spinning at full speed in a microfuge for 30 seconds, and aspriate or pour off growth medium. 5) Thoroughly resuspend the cell pellet in 100µl chilled Solution I using a pipetman. 6) Place the tubes on ice and add 200µl of freshly prepared Solution II. Cap the tube, mix by inversion 8-10 times and return tubes to ice. At this stage the cells will lyse and the solution will grow clear and viscous. 7) Add 150µl of Solution III. Cap tube, mix by inversion 8-10 times and return to ice. The addition of solution III will cause the formation of a flocculent precipitate. 8) Centrifuge for 6 minutes at room temperature at full speed in a microfuge. 9) Transfer the supernatant by pouring to a new microfuge tube. Any visible debris that is transferred can be removed with a toothpick or pipet tip. 10) Precipitate the DNA by adding 1 ml room temperature 100% ethanol and mixing by inversion. 11) Centrifuge for 6 minutes at room temperature in a microfuge. 12) Pour off the supernatant and rinse the pellet by adding 500µl of room temperature 70% ethanol. 13) Pour off the ethanol and drain the tube by resting it upsidedown on a paper towel. Allow to dry completely. 14) Resuspend in 20µl TE. Solution1: 25mM TrisHCl pH 8.0 50mM Glucose 10mM EDTA After cells have been resuspended, add Lysozyme to 2.5mg/ml Solution2: 0.2N NaOH 1% SDS Solution3: 3M Potassium Acetate pH 4.8 This is a tricky solution to prepare. It is made by adding glacial acetic acid to a solution of 5M potassium acetate to achieve a pH of 4.8. This is accomplished by adding a minimal amount of water to the potassium acetate and then adding the acetic acid until the potassium acetate is dissolved and the pH has reached 4.8. Alternatively, the solution can be made by assemblying 60ml 5M KOAce, 11.5 ml glacial acetic acid, and 28.5 ml water.