Basic procedures for bacteria culture-2
E. Elution of DNA fragments from agarose
DNA fragments are eluted from low-melting temperature agarose gels using an unpublished procedure first developed by Dr. Roe. Here, the band of interest is excised with a sterile razor blade, placed in a microcentrifuge tube, frozen at -70degC, and then melted. Then, TE-saturated phenol is added to the melted gel slice, and the mixture again is frozen and then thawed. After this second thawing, the tube is centrifuged and the aqueous layer removed to a new tube. Residual phenol is removed with two ether extractions, and the DNA is concentrated by ethanol precipitation.
Protocol
1. Place excised DNA-containing agarose gel slice in a 1.5 ml microcentrifuge tube and freeze at -70degC for at least 15 minutes, or until frozen. It is possible to pause at this stage in the elution procedure and leave the gel slice frozen at -70degC.
2. Melt the slice by incubating the tube at 65degC.
3. Add one-volume of TE-saturated phenol, vortex for 30 seconds, and freeze the sample at -70degC for 15 minutes.
4. Thaw the sample, and centrifuge in a microcentrifuge at 12,000 rpm for 5 minutes at room temperature to separate the phases. The aqueous phase then is removed to a clean tube, extracted twice with equal volume ether, ethanol precipitated, and the DNA pellet is rinsed and dried.
F. Kinase end-labeling of DNA
Typical 5'-kinase labeling reactions included the DNA to be labeled, [[gamma]]-32-P-rATP, T4 polynucleotide kinase, and buffer (3). After incubation at 37degC, reactions are heat inactivated by incubation at 80degC. Portions of the reactions are mixed with gel loading dye and loaded into a well of a polyacrylamide gel and electrophoresed. The gel percentage and electrophoresis conditions varied depending on the sizes of the DNA molecules of interest. After electrophoresis, the gel is dried and exposed to x-ray film, as discussed below for radiolabeled DNA sequencing.
Protocol
1. Add the following reagents to a 0.5 ml microcentrifuge tube, in the order listed:
sterile ddH2O q.s 10X kinase buffer 1 ul DNA x ul [[gamma]]-[32-P]-rATP 10 uCi T4 polynucleotide kinase 1 ul (3U/ul) 10 ul
[[gamma]]-[32-P]-rATP (35020) ICN and T4 polynucleotide kinase (70031) from United States Biochemicals.
2. Incubate at 37degC for 30-60 minutes.
3. Heat the reaction at 65degC for 10 minutes to inactivate the kinase.
G. Bacterial cell maintenance
Four strains of E. coli are used in these studies: JM101 for M13 infection and isolation (4), XL1BMRF' (Stratagene) for M13 or pUC-based DNA transformation (5), and ED8767 for cosmid DNA transformation (6-8). To maintain their respective F' episomes necessary for M13 viral infection (9), JM101 is streaked onto a M9 minimal media (modified from that given in reference (1) plate and XL1BMRF' is streaked onto an LB plate (1) containing tetracycline. ED8767 is streaked onto an LB plate. These plates are incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid media are inoculated with a smear of several colonies and incubated at 37degC for 8 hours, and those cultures then are transferred into 50 ml of respective liquid media and further incubated 12-16 hours. Glycerol is added to a final concentration of 20%, and the glycerol stock cultures are distributed in 1.3 ml aliquots and frozen at -70degC until use (1).
Protocol
1. Streak a culture of the bacterial cell strain onto an agar plate of the respective medium, listed below, and incubate at 37degC overnight.
E. coli strain Agar Medium/Liquid Media XL1BMRF' (Stratagene) LB-Tet JM101 M9 ED8767 LB
2. Pick several colonies into a 12 X 75 mm Falcon tube containing a 2 ml aliquot of the respective liquid media, and incubate for 8-10 hours at 37degC with shaking at 250 rpm.
3. Transfer the 2 ml culture into an Ehrlenmeyer flask containing 50 ml of the respective liquid media and further incubate overnight (12-16 hours) at 37degC with shaking at 250 rpm.
4. Add 12.5 ml of sterile glycerol for a final concentration of 20%, and distribute the culture in 1.3 ml aliquots into 12 X 75 mm Falcon tubes.
5. Store glycerol cell stocks frozen at -70degC until use.
Notes on Restriction/Modification Bacterial Strains:
1. EcoK (alternate=EcoB)-hsdRMS genes=attack DNA not protected by adenine methylation. (ED8767 is EcoK methylation -). (10)
2. mcrA (modified cytosine restriction), mcrBC, and mrr=methylation requiring systems that attack DNA only when it IS methylated (Ed8767 is mrr+, so methylated adenines will be restricted. Clone can carry methylation activity.) (10)
3. In general, it is best to use a strain lacking Mcr and Mrr systems when cloning genomic DNA from an organism with methylcytosine such as mammals, higher plants , and many prokaryotes.(11)
4. The use of D(mrr-hsd-mcrB) hosts=general methylation tolerance and suitability for clones with N6 methyladenine as well as 5mC (as with bacterial DNAs). (12)
5. XL1-Blue MRF'=D(mcrA)182, D(mcrCB-hsdSMR-mrr)172,endA1, supE44, thi-1, recA, gyrA96, relA1, lac, l-, [F' proAB, lacIqZDM15, Tn10(tetr)].
Host Mutation Descriptions:
ara Inability to utilize arabinose. deoR Regulatory gene that allows for constitutive synthesis for genes involved in deoxyribose synthesis. Allows for the uptake of large plasmids. endA DNA specific endonuclease I. Mutation shown to improve yield and quality of DNA from plasmid minipreps. F' F' episome, male E. coli host. Necessary for M13 infection. galK Inability to utilize galactose. galT Inability to utilize galactose. gyrA Mutation in DNA gyrase. Confers resistance to nalidixic acid. hfl High frequency of lysogeny. Mutation increases lambda lysogeny by inactivating specific protease. lacI Repressor protein of lac operon. LacIq is a mutant lacI that overproduces the repressor protein. lacY Lactose utilization; galactosidase permease (M protein). lacZ b-D-galactosidase; lactose utilization. Cells with lacZ mutations produce white colonies in the presence of X-gal; wild type produce blue colonies. lacZdM15 A specific N-terminal deletion which permits the a-complementation segment present on a phagemid or plasmid vector to make functional lacZ protein. Dlon Deletion of the lon protease. Reduces degradation of b-galactosidase fusion proteins to enhance antibody screening of l libraries. malA Inability to utilize maltose. proAB Mutants require proline for growth in minimal media. recA Gene central to general recombination and DNA repair. Mutation eliminates general recombination and renders bacteria sensitive to UV light. rec BCD Exonuclease V. Mutation in recB or recC reduces general recombination to a hundredth of its normal level and affects DNA repair. relA Relaxed phenotype; permits RNA synthesis in the absence of protein synthesis. rspL 30S ribosomal sub-unit protein S12. Mutation makes cells resistant to streptomycin. Also written strA. recJ Exonuclease involved in alternate recombination pathways of E. coli. strA See rspL. sbcBC Exonuclease I. Permits general recombination in recBC mutants. supE Supressor of amber (UAG) mutations. Some phage require a mutation in this gene in order to grow. supF Supressor of amber (UAG) mutations. Some phage require a mutation in this gene in order to grow. thi-1 Mutants require vitamin B1(thiamine) for growth on minimal media. traD36 mutation inactivates conjugal transfer of F' episome. umuC Component of SOS repair pathway. uvrC Component of UV excision pathway. xylA Inability to utilize xylose. dam DNA adenine methylase/ Mutation blocks methylation of Adenine residues in the recognition sequence 5'-G*ATC-3' (*=methylated) dcm DNA cytosine methylase/Mutation blocks methylation of cytosine residues in the recognition sequences 5'-C*CAGG-3' or 5'-C*CTGG-3' (*=methylated) hsdM E. coli methylase/ Mutation blocks sequence specific methylation AN6*ACNNNNNNGTGC or GCN6*ACNNNNNNGTT (*=methylated). DNA isloated from a HsdM- strain will be restricted by a HsdR+ host. hsd R17 Restriction negative and modification positive. (rK-, mK+) Allows cloning of DNA without cleavage by endogenous restriction endonucleases. DNA prepared from hosts with this marker can efficiently transform rK+ E. coli hosts. hsdS20 Restriction negative and modification negative. (rB-, mB-) Allows cloning of DNA without cleavage by endogenous restriction endonucleases . DNA prepared from hosts with this marker is unmethylated by the hsdS20 modificationsystem. mcrA E. coli restriction system/ Mutation prevents McrA restriction of methylated DNA of sequence 5'-C*CGG (*=methylated). mcrCB E. coli restriction system/ Mutation prevents McrCB restriction of methylated DNA of sequence 5'-G5*C, 5'-G5h*C, or 5'-GN4*C (*=methylated). mrr E. coli restriction system/ Mutation prevents Mrr restriction of methylated DNA of sequence 5'-G*AC or 5'-C*AG (*=methylated). Mutation also prevents McrF restriction of methylated cytosine sequences.
Other Descriptions:
cmr Chloramphenicol resistance kanr Kanamycin resistance tetr Tetracycline resistance strr Streptomycin resistance D Indicates a deletion of genes following it. Tn10 A transposon that normally codes for tetr Tn5 A transposon that normally codes for kanr spi- Refers to red-gam- mutant derivatives of lambda defined by their ability to form plaques on E. coli P2 lysogens. Commonly used bacterial strains C600 - F-, e14, mcrA, thr-1 supE44, thi-1, leuB6, lacY1, tonA21, l- -for plating lambda (gt10) libraries, grows well in L broth, 2x TY, plate on NZYDT+Mg. -Huynh, Young, and Davis (1985) DNA Cloning, Vol. 1, 56-110. DH1 - F-, recA1, endA1, gyrA96, thi-1, hsdR17 (rk-, mk+), supE44, relA1, l- -for plasmid transformation, grows well on L broth and plates. -Hanahan (1983) J. Mol. Biol. 166, 557-580. XL1Blue-MRF' - D(mcrA)182, D(mcrCB-hsdSMR-mrr)172,endA1, supE44, thi-1, recA, gyrA96, relA1, lac, l-, [F'proAB, lac IqZDM15, Tn10 (tetr)] -For plating or glycerol stocks, grow in LB with 20 mg/ml of tetracycline. For transfection, grow in tryptone broth containing 10 mM MgSO4 and 0.2% maltose. (No antibiotic--Mg++ interferes with tetracycline action.) For picking plaques, grow glycerol stock in LB to an O.D. of 0.5 at 600 nm (2.5 hours?). When at 0.5, add MgSO4 to a final concentration of 10 mM. SURE Cells - Stratagene - e14(mcrA), D(mcrCB- hsdSMR-mrr)171, sbcC, recB, recJ, umuC::Tn5 (kanr), uvrC, supE44, lac, gyrA96, relA1, thi-1, end A1[F'proAB, lacIqDM15, Tn10(tetr)]. An uncharacterized mutation enhances the a - complementation to give a more intense blue color on plates containing X-gal and IPTG. GM272 - F-, hsdR544 (rk-, mk-), supE44, supF58, lacY1 or 苐acIZY6, galK2, galT22, metB1m, trpR55, l- -for plasmid transformation, grows well in 2x TY, TYE, L broth and plates. -Hanahan (1983) J. Mol. Biol. 166, 557-580. HB101 - F-, hsdS20 (rb-, mb-), supE44, ara14, galK2, lacY1, proA2, rpsL20 (strR), xyl-5, mtl-1, l-, recA13, mcrA(+), mcrB(-) -for plasmid transformation, grows well in 2x TY, TYE, L broth and plates. -Raleigh and Wilson (1986) Proc. Natl. Acad. Sci. USA 83, 9070-9074. JM101 - supE, thi, ?lac-proAB), [F', traD36, proAB, lacIqZ芃15], restriction: (rk+, mk+), mcrA+ -for M13 transformation, grow on minimal medium to maintain F episome, grows well in 2x TY, plate on TY or lambda agar. -Yanisch-Perron et al. (1985) Gene 33, 103-119. XL-1 blue recA1, endA1, gyrA96, thi, hsdR17 (rk+, mk+), supE44, relA1, l-, lac, [F', proAB, lacIqZ芃15, Tn10 (tetR)] -for M13 and plasmid transformation, grow in 2x TY + 10 礸/ml Tet, plate on TY agar + 10 礸/ml Tet (Tet maintains F episome). -Bullock, et al. (1987) BioTechniques 5, 376-379. GM2929 - from B. Bachman, Yale E.coli Genetic Stock Center (CSGC#7080); M.Marinus strain; sex F-; (ara-14, leuB6, fhuA13, lacY1, tsx-78, supE44, [glnV44], galK2, galT22, l-, mcrA, dcm-6, hisG4,[Oc], rfbD1, rpsL136, dam-13::Tn9, xyl-5, mtl-1, recF143, thi-1, mcrB, hsdR2.) MC1000 - (araD139, D[ara-leu]7679, galU, galK, D[lac]174, rpsL, thi-1). obtained from the McCarthy lab at the University of Oklahoma. ED8767 (F-,e14-[mcrA],supE44,supF58,hsdS3[rB-mB-], recA56, galK2,galT22,metB1, lac-3 or lac3Y1 - obtained from Nora Heisterkamp and used as the host for abl and bcr cosmids.
H. Fragment purification on Sephacryl S-500 spin columns
DNA fragments larger than a few hundred base pairs can be separated from smaller fragments by chromatography on a size exclusion column such as Sephacryl S-500. To simplify this procedure, the following mini-spin column method has been developed.
1. Thoroughly mix a fresh, new bottle of Sephacryl S-500, distribute in 10 ml portions, and store in screw cap bottles or centrifuge tubes in the cold room.
2. Prior to use, briefly vortex the matrix and without allowing to settle, add 500 ul of this slurry to a mini-spin column (Millipore) which has been inserted into a 1.5 ml microcentrifuge tube.
3. Following centrifugation at 2K RPM in a table top centrifuge, carefully add 200 ul of 100 mM Tris-HCl (pH 8.0) to the top of the Sephacryl matrix and centrifuge for 2 min. at 2K RPM. Repeat this step twice more. Place the Sephacryl matrix-containing spin column in a new microcentrifuge tube.
4. Then, carefully add 40 ul of nebulized cosmid, plasmid or P1 DNA which has been end repaired to the Sephacryl matrix (saving 2 ul for later agarose gel analysis) and centrifuge at 2K RPM for 5 minutes. Remove the column, save the solution containing the eluted, large DNA fragments (fraction 1). Apply 40 ul of 1xTM buffer and recentrifuge for 2 minutes at 2K RPM to obtain fraction 2 and repeat this 1xTM rinse step twice more to obtain fractions 3 and 4.
5. To check the DNA fragment sizes, load 3-5 ul of each eluant fraction onto a 0.7% agarose gel that includes as controls, 1-2 ul of a PhiX174-HaeIII digest and 2 ul of unfractionated, nebulized DNA saved from step 4 above.
6. The fractions containing the nebulized DNA in the desired size ranges (typically fractions 1 and 2) are separately phenol extracted and concentrated by ethanol precipitation prior to the kinase reaction.
