This is a cached page for the URL (http://www.tree.caltech.edu/protocols/BAC_lib_construction.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache

How to build a BAC library:

(Ung-Jin Kim)

Contents

Introduction

    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

BAC LIGATION

    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.