Agl1农杆菌感受态细胞使用说明书
感受态细胞手册
NovagenUSA and Canada Europe All Other CountriesTel (800) 526-7319 novatech@FranceFreephone0800 126 461GermanyFreecall0800 100 3496IrelandToll Free1800 409 445United KingdomFreephone0800 622 935All otherEuropean Countries+44 115 943 0840Contact Your Local Distributornovatech@Competent CellsTable of ContentsAbout the Kits (2)Des ription 2Components 2Storage 2Strain information 3Transformation Protocol for Experienced Users (7)Transformation - Detailed Protocol (8)Handling tips 8Pro edure 9Plating Techniques 10Troubleshooting (12)Ordering Information (13)Strain Genotypes (16)Genetic Marker Descriptions (18)References (19)Academic and Non-profit Laboratory Assurance Letter (20)Bacterial Strain Non-distribution Agreement (21)© 2009 EMD Chemicals Inc., an affiliate of Merck KGaA, Darmstadt, Germany. All rights reserved. Novagen®, the Novagen® logo, and Perfectly Blunt® are registered trademarks of EMD Chemicals Inc. in the United States and in certain other jurisdictions. A ccep T or™, ColiRollers™, Origami™, pETBlue™, pETcoco™, Rosetta™, RosettaBlue™, Rosetta-gami™, Singles™, TriEx™ , and Tuner™ are trademarks of EMD Chemicals Inc.The T7 expression system host strains (DE3) are covered under U.S. Patents 4,952,496, 5,693,489, and 5,896,320. For academic and non-profit laboratories, a non-distribution license is included with the product. Commercial customers must obtain a research-use license from Brookhaven Science Associates prior to purchase.Origami™ 2, Origami B, Rosetta™, Rosetta 2, Rosetta-gami™ 2, and Rosetta-gami B strains are proprietary strains of EMD Chemicals Inc. A non-distribution license is included in this user protocol.About the KitsDescriptionNovagen Competent Cells enable convenient, efficient construction of plasmid recombinants. Thecells are grown and made chemically competent by an optimized procedure, followed byverification of cloning efficiency and strain identity. The cells in the standard kits are provided asfrozen 0.2-ml aliquots; each vial can be used for 10 transformations. The cells in the Singles™ kitsare provided as single-use 50-µl aliquots for greater efficiency and convenience, and are packed inkits for either 11 or 22 transformations. Reproducible high efficiencies are available in a variety ofE. coli strains, including NovaBlue for routine cloning, blue/white screening, and plasmidpreparation, as well as T7 expression system strains (λDE3 lysogens), and isogenic control strains(non-lysogens) for superior performance in protein expression applications. The designation (DE3)indicates that the host is a lysogen of λDE3, and therefore carries a chromosomal copy of theT7 RNA polymerase gene under control of the lacUV5 promoter (1–3). Such strains are suitable forproduction of protein from target genes cloned in pET vectors or other T7-driven expressionvectors. pLysS is a designation given to hosts carrying a chloramphenicol-resistant plasmid with aP15A replicon that encodes T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase (4–5).This strain is used to suppress basal expression of T7 RNA polymerase prior to induction and thusstabilize pET recombinants encoding target proteins that affect cell growth and viability. The pLacIdesignation is given to hosts carrying a compatible plasmid that encodes lac repressor undercontrol of its wild type promoter. Expression hosts carrying this plasmid were specificallydesigned for compatibility with the pETBlue™ and pTriEx™ (1.1–4) plasmid series. The λDE3Lysogenization Kit is also available for making new expression hosts with other geneticbackgrounds.ComponentsStandard 0.4 ml and 1 ml Kits• 2 or 5 × 0.2 ml Competent Cells• 2 or 4 × 2 ml SOC Medium•10 µl Test Plasmid (ampicillin resistant)Singles Kits•11 or 22 × 50 µl Competent Cells• 2 or 4 × 2 ml SOC Medium•10 µl Test Plasmid (ampicillin resistant)StorageStore all components at –70°C or below.USA and Canada Germany United Kingdom and Ireland All Other CountriesUSA and Canada GermanyUnited Kingdom and Ireland All Other Countries Strain informationStrains offered as competent cells are listed in the table below. Genotypes are found on page 16. Strains having the designation (DE3) are lysogenic for a λ prophage that contains an IPTG-inducible T7 RNA polymerase. λDE3 lysogens are designed for protein expression from pET,pETcoco™, pETBlue™, pTriEx™, pCDF, pRSF, and Duet vectors. Strains having the pLysS designation carry a pACYC184-derived plasmid that encodes T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase that serves to repress basal expression of target genes under the control of the T7 promoter. Strains having the designation pLacI carry a pACYC-derived plasmid that encodes the lac repressor, which serves to suppress basal expression of target genes under T7 promoter control in pETBlue and pTriEx (1.1–4) plasmids. All Rosetta™ and Rosetta 2 strains contain pRARE or pRARE2, respectively, a pACYC184-derived plasmid.(continued on next page; see footnotes on page 6)DescriptionStrains Resistance 1Derivation Key Feature(s)B834 is the parental strain for BL21 (6). These hosts are methionine auxotrophs and allow high specific activitylabeling of target proteins with 35S-methionine and selenomethionine for crystallography (7). This strain is alsodeficient in the lon (8) and ompT proteases.B834(DE3) B834(DE3)pLysSnone CamB strain Met auxotroph; 35S-met labeling BL21 is the most widely used host background for protein expression and has the advantage of being deficient in the lon (8) and ompT proteases.BL21 BL21(DE3) BL21(DE3)pLysS none none Cam B834 lon and ompT protease deficient BLR is a recA derivative of BL21 (9) that improves plasmid monomer yields and may help stabilize targetplasmids containing repetitive sequences or whose products may cause the loss of the DE3 prophage (10). These strains are also deficient in the lon and ompT proteases. BLR(DE3)BLR(DE3)pLysSTetTet + CamBL21 BL21 recA mutant; stabilizes tandemrepeatsHMS174 strains provide the recA mutation in a K-12 background. Like BLR, these strains may stabilize certain target genes whose products may cause the loss of the DE3 prophage. HMS174HMS174(DE3)HMS174(DE3)pLysSRif RifRif + CamK-12recA mutant, Rif resistanceNovaBlue is a K-12 strain ideally suited as an initial cloning host due to its high transformation efficiency,blue/white screening capability (with appropriate plasmids) and recA endA mutations, which result in high yieldsof excellent quality plasmid DNA. The NovaBlue T1Rstrain has the added benefit of resistance to T1 and T5 phage. The DE3 lysogen of NovaBlue is potentially useful as a stringent host due to the presence of the lacI q repressor encoded by the F episome. Note, however that the DE3 lysogen cannot be used for blue/white screening of recombinant plasmids. NovaBlueNovaBlue(DE3)Tet TetK-12recA -, endA -, lacI q ; recommended for cloning, plasmid preps (non-DE3 only)NovaBlue T1R TetK-12 recA -, endA -, lacI q ; tonArecommended for cloning, plasmid preps; resistant to T1 and T5 phageUSA and Canada GermanyUnited Kingdom and Ireland All Other Countries DescriptionStrains Resistance 1Derivation Key Feature(s)NovaF – is a K-12 strain ideally suited as a cloning host due to its high transformation efficiency and recAendA mutations, which result in high yields of excellent quality plasmid DNA. In contrast to NovaBlue, NovaF – lacks the F episome that encodes lacZ ∆M15 and lacI q mutations. Therefore this strain is notappropriate for blue/white screening by α-complementation or for propagation of expression vectors that contain an E. coli promoter controlled by a lac operator sequence, i.e., tac , trc , T5, etc. in the absence of an additional source of lac repressor. NovaF – is recommended for the preparation of pETcoco™ recombinants (review User Protocol TB333).NovaF –none K-12 recA -, endA -; recommended forcloning, plasmid preps Origami™ 2 host strains are K-12 derivatives that have mutations in both the thioredoxin reductase (trxB ) and glutathione reductase (gor ) genes, which greatly enhances disulfide bond formation in the cytoplasm (11). Unlike the original Origami strains, the Origami 2 strains are kanamycin sensitive, making these host strains compatible with many Novagen expression vectors. The gor mutation is still selected for by tetracycline, as are the original strains. Origami 2Origami 2(DE3)Origami 2(DE3)pLysS Origami 2(DE3)pLacI 2Tet + Str 3 Tet + Str 3Tet + Str 3 + Cam Tet + Str 3 + CamK-12 Kan sensitive, trxB/gor mutant,greatly facilitates cytoplasmic disulfide bond formation, Leu auxotroph,Origami B host strains carry the same trxB/gor mutations as the original Origami strain, except that they are derived from a lacZY mutant of BL21. Thus the Origami B strains combine the desirable characteristics of BL21, Tuner™, and Origami hosts in one strain background. The trxB and gor mutations are selectable on kanamycin and tetracycline, respectively; therefore, these strains are not compatible with kanamycin- or tetracycline-resistant plasmids. Origami BOrigami B(DE3)Origami B(DE3)pLysS Origami B(DE3)pLacI 2Kan + Tet Kan + TetKan + Tet + Cam Kan + Tet + CamTuner™ (B strain)trxB /gor mutant, greatlyfacilitates cytoplasmic disulfide bond formationBL21 lacZY deletion mutant;allows precise control with IPTGRosetta™ and Rosetta 2 host strains are BL21 derivatives designed to enhance the expression ofeukaryotic proteins that contain codons rarely used in E. coli . (13–17). The original Rosetta strains supply tRNAs for the codons AUA, AGG, AGA, CUA, CCC, and GGA on a compatible chloramphenicol-resistantplasmid, pRARE (18). The Rosetta 2 strains supply a seventh rare codon (CGG) in addition to the six found in the original Rosetta strains (19). By supplying rare codons, the Rosetta strains provide for “universal”translation, where translation would otherwise be limited by the codon usage of E. coli . (15, 16, 20, 21). The tRNA genes are driven by their native promoters (18). In the pLysS and pLacI derivatives of these strains, the rare tRNA genes are present on the same plasmids that carry the T7 lysozyme and lac repressor genes, respectively. RosettaRosetta(DE3)Rosetta(DE3)pLysS Rosetta(DE3)pLacI 2 Cam Cam Cam Cam BL21Expresses six rare tRNAs; facilitates expression of genes that encode rare E. coli codons Rosetta 2Rosetta 2(DE3)Rosetta 2(DE3)pLysS Rosetta 2(DE3)pLacI 2Cam Cam Cam CamBL21Expresses seven rare tRNAs; facilitates expression of genes that encode rare E. coli codons(continued on next page; see footnotes on page 6)DescriptionStrains Resistance1 DerivationKeyFeature(s) Rosetta-gami™ 2 host strains combine features of Origami™ 2 and Rosetta™ 2, allowing for enhanced disulfide bond formation and enhanced expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains are derived from Origami 2, a kanamycin-sensitive K-12 strain carrying the trxB and gor mutations for disulfide bonds formation in the cytoplasm. The cells carry the chloramphenicol-resistant plasmid, pRARE2, which supplies tRNAs for seven rare codons, AUA, AGG, AGA, CUA, CCC, GGA, and CGG under the control of their native promoter. The gor mutation is selectable on tetracycline.Rosetta-gami 2Rosetta-gami 2(DE3) Rosetta-gami 2(DE3)pLysS Rosetta-gami 2(DE3)pLacI2Tet + Str3 + CamTet + Str3 + CamTet + Str3 + CamTet + Str3 + CamOrigami 2(K-12)Expresses seven rare tRNAs;facilitates expression of genesthat encode rare E. coli codonsKan sensitive, trxB/gor mutant,greatly facilitates cytoplasmicdisulfide bond formation, LeuauxotrophRosetta-gami B strains combine the key features of BL21 (and its Tuner™ derivative), Origami, and Rosetta to enhance both the expression of eukaryotic proteins and the formation of target protein disulfide bonds in the bacterial cytoplasm. These strains are compatible with ampicillin- or spectinomycin-resistant vectors.Rosetta-gami BRosetta-gami B(DE3) Rosetta-gami B(DE3)pLysS Rosetta-gami B(DE3)pLacI2Origami B(B strain)Expresses six rare tRNAs;facilitates expression of genesthat encode rare E. coli codonstrxB/gor mutant, greatlyfacilitates cytoplasmic disulfidebond formationBL21 lacZY deletion mutant;allows precise control with IPTGRosettaBlue™ host strains are NovaBlue derivatives that combine high transformation efficiency and recA endA lacI q mutations with enhanced expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains supply tRNAS for AGG, AGA, AUA, CUA, CCC, and GGA on a compatible chloramphenicol-resistant plasmid. In RosettaBlue(DE3)pLysS and RosettaBlue(DE3)pLacI, the rare tRNA genes are present on the same plasmids that carry the T7 lysozyme and lac repressor genes, respectively. Blue/white screening is not possible with RosettaBlue(DE3) strains due to the presence of the lacZα-peptide coding sequence in the DE3 lysogenic phage.RosettaBlue™ RosettaBlue(DE3) RosettaBlue(DE3)pLysS RosettaBlue(DE3)pLacI2NovaBlue(K-12)Expresses rare tRNAs; facilitatesexpression of genes that encoderare E. coli codonsrecA-, endA-, lacI q; hightransformation efficiencyTuner™ strains are lacZY deletion mutants of BL21, which enable adjustable levels of protein expression throughout all cells in a culture. The lac permease (lacY) mutation allows uniform entry of IPTG into all cells in the population. Unlike lactose (or arabinose), IPTG is a gratuitous inducer that can enter E. coli cells independently from permease pathways. This allows induction with IPTG to occur in a true concentration-dependent fashion that is exceptionally uniform throughout the culture. By adjusting the concentration of IPTG, expression can be regulated from very low expression levels up to the robust, fully induced expression levels commonly associated with pET vectors. Lower level expression may enhance the solubility and activity of difficult target proteins. These strains are also deficient in the lon and ompT proteases.Tuner™Tuner(DE3) Tuner(DE3)pLysS Tuner(DE3)pLacI2BL21 BL21lacZY deletion mutant;allows precise control with IPTG(see footnotes on page 6)USA and Canada Germany United Kingdom and Ireland All Other Countries1.The Resistance column in the table refers to selectable resistant marker(s) possessed by the strain in the absenceof target plasmids. Appropriate concentrations for selection are as follows:Kan: 15 µg/ml kanamycinCam: 34 µg/ml chloramphenicolTet : 12.5 µg/ml tetracyclineRif: 200 µg/ml rifampicinStr: 50 µg/ml streptomycin2.Strains with the pLacI plasmid are appropriate hosts for pTriEx™ (1.1–4) and pETBlue™ vectors only.3.These strains carry a mutation in ribosomal protein (rpsL) conferring resistance to streptomycin; thereforestreptomycin is not necessary to maintain strain genotype. If using pCDF vectors, spectinomycin must be used forantibiotic selection because rpsL mutation confers streptomycin resistance.Antibiotics/IPTG available separately Size Cat. No.69101-3 Carbenicillin 5g220551gChloramphenicol 25Kanamycin Sulfate 5 g 420311Tetracycline Hydrochloride 10 g 58346Streptomycin Sulfate, Streptomyces sp. 100 g 5711Spectinomycin, Dihydrochloride,Pentahydrate, Streptomyces sp. 10 g 567570100 mM IPTG Solution 10 × 1.5 ml 70527-3X-Gal Solution, 40 mg/ml in DMSO 3 × 1.0 ml 71077-3USA and Canada Germany United Kingdom and Ireland All Other CountriesUSA and Canada Germany United Kingdom and Ireland All Other CountriesTransformation - Detailed ProtocolNote: When selecting for the expression of β-lactamase, the antibiotic carbenicillin is recommendedinstead of ampicillin. Carbenicillin is less sensitive to the drop in the pH of the growth medium thattypically accompanies bacterial growth.Competent cells in the standard kits are provided in 0.2-ml aliquots. The standard transformationreaction uses 20 µl cells, so each tube contains enough cells for 10 transformations. Singles™competent cells are provided in 50-µl aliquots, which are used “as is” for single 50-µltransformations. Please note that there are a few steps in the protocol that vary for the Singles vs.standard kits.DNA in ligation reactions containing high-quality reagents is suitable for direct addition to Novagencompetent cells. Inactivation of the ligase is not required prior to transformation. Fortransformation, 1 µl of the ligation reaction usually yields sufficient numbers of colonies forscreening. Up to 5 µl of the ligation reaction containing high-quality reagents can be added toSingles Competent Cells without reducing transformation efficiency.Plasmid DNA isolated using standard miniprep procedures is also usually satisfactory; however, formaximum efficiency, the sample DNA should be free of phenol, ethanol, salts, protein, anddetergents, and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) or in water.Transformation efficiencies will generally be 10- to 100-fold higher with supercoiled plasmids thanwith ligation reactions, so it is often necessary to dilute standard plasmid preparations in TE bufferor water prior to transformation. One microliter containing 1 to 10 ng plasmid DNA is usuallysufficient to produce hundreds of colonies. For cotransformations into expression strains usingtwo supercoiled plasmids, add 1 µl containing 10–40 ng of each plasmid into expression straincompetent cells. Note that a higher concentration of DNA will yield a higher number oftransformants on the plate, but the transformation efficiency of the cells will decrease.Handling tips1.Upon receipt from Novagen, verify that the competent cells are still frozen and that dry ice isstill present in the shipping container. Immediately place the competent cells at –70°C orbelow. For optimal results, do not allow the cells to thaw at any time prior to use.2.Handle only the rim of the tube and the tube cap to prevent the cells from warming. Keep thecells on ice whenever possible.3.To mix cells, finger-flick the tube 1–3 times. NEVER vortex the competent cells.4.Cells can be refrozen at –70°C and used at a later date; however, transformation efficienciesmay decline several-fold with each freeze-thaw cycle. To avoid multiple freeze-thaw cycles ofthe standard cells, dispense the cells into aliquots after the initial thaw and store the aliquotsat –70°C or below (note that Singles cells are provided as 50-µl aliquots, which are used “as is”and should not be divided). To dispense aliquots of cells from the 0.2 ml stock, remove thestock tube quickly from the ice and finger-flick 1–2 times to mix prior to opening the tube.Remove a 20-µl aliquot from the middle of the cells, and replace the tube immediately on ice.Place the aliquot immediately into the bottom of a pre-chilled 1.5-ml tube, mix by pipettingonce up and down, and then immediately close the tube and replace on ice. After all of thealiquots have been removed, return any unused tubes to the freezer before proceeding with thetransformation.USA and Canada Germany United Kingdom and Ireland All Other CountriesProcedure1.Remove the appropriate number of competent cell tubes from the freezer (include one extrasample for the Test Plasmid positive control, if desired). Immediately place the tubes on ice, so that all but the cap is immersed in ice. Allow the cells to thaw on ice for 2–5 min.2.Visually examine the cells to see that they have thawed and gently finger-flick the tube 1–2times to evenly resuspend the cells. The cells are then ready for removal of an aliquot(Standard Kits), or for the addition of the DNA (Singles Kits).3.Standard Kits:Place the required number of 1.5-ml snap-cap polypropylene tubes on ice to pre-chill. Pipet 20 µl aliquots of cells into thepre-chilled tubes. Singles Kits:Proceed to Step 4 or 5, depending on whether a Test Plasmid sample is included as a positive control.4.(Optional) To determine transformation efficiency, add 1 µl (0.2 ng) Test Plasmid to one of thetubes containing cells. Stir gently to mix and return the tube to the ice.5.Add 1 µl of a ligation reaction or purified plasmid DNA (1-10 ng/ µl plasmid) directly to thecells. Stir gently to mix and return the tube to the ice, making sure that the tube is immersed inice except for the cap. Repeat for additional samples.Note: Transformation efficiencies can be increased several fold by diluting the ligation reaction 5-fold with TE or water prior to adding the DNA to the cells, or by extracting the ligation reaction twicewith 1:1 TE-buffered phenol:CIAA (24:1 chloroform:isoamyl alcohol), once with CIAA, precipitatingin the presence of sodium acetate, and resuspending in TE or water before adding the DNA to thecells.6.Incubate the tubes on ice for 5 min.7.Heat the tubes for exactly 30 s in a 42°C water bath; do not shake.Note: This “heat shock” step is most easily accomplished if the tubes are in a rack that leaves the lower halves of the tubes exposed. Hold the rack in the water bath so that the lower halves of the tubesare submerged for 30 s, and then replace the rack on ice.8.Place the tubes on ice for 2 min.9.Standard Kits:Add 80 µl of room temperature SOCmedium to each tube. Keep the tubes onice until all have received SOC. Singles Kits:Add 250 µl of room temperature SOC medium to each tube. Keep the tubes on ice until all have received SOC.Selection for transformants is accomplished by plating on medium containing antibiotic(s) for theplasmid-encoded drug resistance(s). Additional host-specific antibiotics also may be appropriate toinsure maintenance of the host encoded feature(s) (see chart beginning on page 3).When using strains other than NovaBlue: incubate at 37°C while shaking at 250 rpm for60 min prior to plating on selective media.When using NovaBlue: if selecting for ampicillin or chloramphenicol resistance, plate 5–50 µlcells directly on selective media (plus IPTG/X-gal for plasmids which permit blue/whitescreening). If selecting for kanamycin or streptomycin/spectinomycin resistance, shake at37°C (250 rpm) for 30 min prior to plating on selective media.Notes: The outgrowth incubation is conveniently performed in a shaking incubator using a test tube rack anchored to the shaking platform. Place each transformation tube in an empty 13 mm × 100 mmglass test tube in the rack. The snap-caps on the transformation tubes prevent them from falling tothe bottom of the test tubes, and all transformation tubes remain vertical.During the outgrowth (or earlier if omitting outgrowth), place the plates at 37°C. If the platescontain a lot of moisture, place them cover-side up and open the cover ~1/3 of the way to allowthe plates to dry for 30–45 min. If the plates do not need drying, keep them closed and place themcover-side down in the 37°C incubator for ~20 min prior to plating.USA and Canada Germany United Kingdom and Ireland All Other CountriesUSA and Canada GermanyUnited Kingdom and Ireland All Other Countries 10. Refer to “Plating Techniques” in the subsequent section for specific instructions. Spread5–50 µl of each transformation on LB agar plates containing the appropriate antibiotic(s) for the plasmid and host strain. When plating less than 25 µl, first pipet a “pool” of SOC onto the plate and then pipet the cells into the SOC. Please see the next section for additional details on plating technique.Important:The appropriate amount of transformation mixture to plate varies with the efficiency of both theligation and the competent cells. For recombinants in NovaBlue, expect 10 5–10 7transformants/µg plasmid, depending on the particular insert and the ligation efficiency.Transformations with the pETcoco™ plasmid require a plating volume of 50 µl to obtain sufficient colonies because the pETcoco plasmid is large (12,272 bp).When using the Test Plasmid, plate no more than 5 µl of the final NovaBlue transformation mix or plate 10 µl of any strain with a 2 × 10 6 efficiency in a pool of SOC on an LB agar plate containing 50 µg/ml carbenicillin or ampicillin (because the Test Plasmid carries the ampicillin resistance gene, bla ).For blue/white screening of recombinants, also include IPTG and X-gal in the LB agar. These can be pre-spread on the plates and allowed to soak in for about 30 min prior to plating. Use 35 µl of 50 mg/ml X-gal in dimethyl formamide and 20 µl 100 mM IPTG (in water) per 82 mm plate.Alternatively, X-gal and IPTG can be added to the LB agar at a final concentration of 70 µg/ml and 80 µM, respectively, just prior to pouring the plates.11. Set the plates on the bench for several min to allow excess liquid to be absorbed, and theninvert and incubate overnight at 37°C.Plating Techniques1. Remove the plates from the incubator. If plating less than 25 µl of the transformation, werecommend plating onto a pool of SOC, which facilitates even colony distribution on the plate surface. Using a sterile pipet tip, place 40–60 µl of SOC in the center of a plate for a plating cushion. 2. To remove the transformation sample, finger-flick the transformation tube 5–8 times, open thecap and immediately remove the sample volume from the middle of the transformation reaction. 3. Transfer the sample to the plate by dispensing the sample volume into the SOC cushion. Afterthe sample is expelled, use the same tip to pipet up the same volume of SOC from the cushion edge and dispense the fluid back into the cushion. (This effectively rinses out your pipet tip.)Plating with ColiRollers™ Plating BeadsTo use ColiRollers, simply dispense 10–20 beads per plate. Cover the plate with its lid and rock the plate back and forth several times. The rolling action of the beads distributes the cells. Several plates can be stacked and shaken at the same time. After all plates have been spread, discard the ColiRollers and incubate (step 4 below).Plating with a standard spreader1. Completely immerse the plating spreader (bent glass rod or equivalent) into ethanol and flameto sterilize. After the flame is extinguished, allow the spreader to cool ~10 s prior to placing the spreader on the plate. To further cool the spreader before spreading the cells, place the spreader on the LB agar at the outside of the plate (not touching the pool of cells). 2. Slowly rotate the plate while supporting the weight of the spreader.Important:Do not press down on the spreader – use just enough contact to spread the cells.3. Spread until the sample is evenly distributed on the plate. If the plates are fairly dry, thesample and cushion will quickly absorb into the plate. If the plates are wet, spread until the sample is evenly distributed. Do not continue to spread until the sample and cushion have absorbed completely into the plate, as overspreading is lethal to the cells. Instead, afterspreading briefly, set the plates upright at room temperature for ~15 min prior to placing them inverted in the 37ºC incubator. This will allow excess moisture to absorb into the plates.ColiRollers Plating Beads (Cat. No. 71013-3) are sterile glass beads that eliminate the use of the spreader and alcohol flame while evenlydistributing cells without damaging the cells.。
TG1感受态细胞说明书
TG1感受态细胞说明书一、简介大肠杆菌经Ca离子处理后可摄取外源DNA（Plasmid、Phage DNA），处于这种状态的细胞称作感受态细胞（Competent Cells）。
在进行基因重组实验时，使用感受态细胞的转化实验应用十分广泛。
制作基因文库、重组质粒体以及进行亚克隆实验时，特别是在目的基因含量十分低的情况时，使用高效的感受态细胞十分重要。
而TG1菌株常常用于噬菌体的制备。
本公司生产的感受态细胞是采用氯化钙处理得到的感受态细胞，使用pUC19质粒检测，转化效率可达107-108，在-70℃下保存几个月转化效率不发生改变。
二、使用方法（1）取出感受态细胞，置于冰上5min，解冻。
（2）然后将连接体系（5~10ul）加入感受态中（～100ul），轻弹感受态离心管底部，混匀，冰浴30分钟。
（3）将离心管放入42℃水浴90秒后，迅速放于冰上。
（4）每管中加500μl LB(SOC)培养基(不含抗生素)，37℃振荡培养1小时。
目的是使质粒上相关的抗性标记基因表达，使菌体复苏。
（5）室温4,500rpm离心5分钟，弃去900μl上清后，用剩余100μl 培养基重悬细胞并涂布到含Amp的LB琼脂平板或含X-gal、IPTG、Amp的LB琼脂平板表面。
（6）将平板置于室温直至液体被吸收。
（7）倒置平皿，于37℃培养，12~16小时后可出现菌落。
三、注意事项１. 用此感受态进行转化需要热激，不能电转化，请勿分装。
2 . 感受态细胞应保存在-70 ℃，不可多次冻融和放置时间过长，以避免降低感受态细胞的转化效率。
3 . 为防止转化实验不成功，可以保留部分连接反应液，以重新转化，将损失降到最低。
4 ．所有操作均需注意无菌操作。
实验三农杆菌感受态细胞制备及转化
农杆菌介导基因转化
农杆菌是普遍存在于土壤中的一种革兰氏阴性细菌，它能在自然条 件下趋化性地感染大多数双子叶植物的受伤部位，并诱导产生冠瘿瘤或发 状根。 根癌农杆菌和发根农杆菌中细胞中分别含有Ti质粒和Ri质粒，其上有 一段T-DNA，农杆菌通过侵染植物伤口进入细胞后，可将T-DNA插入到 植物基因组中,并且可以通过减数分裂稳定的遗传给后代，这一特性成为农 杆菌介导法植物转基因的理论基础。人们将目的基因插入到经过改造的T-
农杆菌的转化加入1mlyep培养基加入1mlyep培养基28慢摇培养24h28慢摇培养24h液氮中速冻1min迅速置于37水浴中5min取100?l农杆菌感受态加入约051?g质粒dna混匀后冰浴30min均匀涂布在含rif和kan的yep平板上28c温育48h至菌落出现离心去上清将细胞重悬于200?lyep培养基中载体分子带有抗生素抗性基因kan感受态受体细胞本身不具有抗生素的抗性没有导入载体的细胞在抗生素培养基上便不能生长
农杆菌感受态的制备
A • 挑取农杆菌单菌落于液体培养基中，28 °C，200 rpm振荡培养过夜活化
B
• 菌液按1:50的比例转接于50ml新鲜培养基中，振荡培养至OD600为0.4-0.6；
C
• 菌液转移至10 ml的离心管中，冰浴30 min
D
• 4℃，5 000 rpm离心5 min, 弃上清，收集菌体
DNA区，借助农杆菌的感染实现外源基因向植物细胞的转移与整合，然
后通过细胞和组织培养技术，再生出转基因植株。 特点：操作简便、成本低、转化率高，广泛应用于双子叶植物的遗传转化。
农杆菌感受态
感受态细胞（competent cell)是细菌细胞具有的能够接受外源DNA
的一种特殊生理状态. 重组DNA要导入宿主细胞，其生理状态很重要，需要将细胞进行特 殊处理，使细胞膜透性发生暂时性改变，成为容易接受外源DNA分 子的状态，即成为感受态细胞。 电击法， CaCl2法。 不同的转化方法需制备不同的感受态。 CaCl2法制备感受态：正在生长的农杆菌细胞加入到低渗的CaCl2溶 液中，0℃下处理便会使细菌细胞膜的透性发生改变，此时的细胞 呈现出感受态.
Stable 电击感受态细胞使用说明
Stable 电击感受态细胞Stable Electroporation Competent CellStable电击感受态细胞基因型：F' proA+B+ lacIq ∆(lacZ)M15 zzf::Tn10 (TetR) ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14- Φ80dlacZ∆M15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 ∆(mrr-hsdRMS -mcrBC)Stable电击感受态细胞说明：Stable电击感受态细胞只能用于电击转化，不可用于热激转化。
Stable菌株是NEB公司开发的高转化效率菌株，是逆转录病毒/慢病毒载体系统推荐使用的菌株，具有与Stbl2，Stbl3完全不同的基因型，但是表现出比Stbl2，Stbl3更优异的性能，特别适合慢病毒或具有末端重复序列DNA片段的克隆。
基因组含有重组酶recA1 rel A1突变，可有效抑制长片段末端重复区的重组，降低错误重组的概率；同时含有核酸酶endA1突变，避免了提取质粒过程中核酸酶的污染，大大提高了高纯度病毒质粒的产量和质量。
lacZΔM15的存在使Stable可用于蓝、白斑筛选，此菌株具有四环素和链霉素抗性，唯地生物开发的Stable电击感受态细胞适用于大质粒的构建或者各种具有末端重复序列的复杂DNA文库构建，经特殊工艺制作，pUC19质粒检测转化效率>2×1010 cfu/μg DNA。
Stable电击感受态细胞操作方法：1. 0.1cm 电击杯和杯盖从储存液中拿出倒置于干净的吸水纸上5分钟，待其沥干水分，正置5分钟，使乙醇充分挥发，待乙醇挥发干净立即插入冰中，压实冰面，电极杯顶离冰面0.5 cm 以方便盖上杯盖，冰中静置5分钟充分降温。
2. 取-80℃保存的Stable电击感受态细胞插入冰中5 分钟，待其融化，加入目的DNA (质粒或连接产物)并用手拨打EP管底轻轻混匀，避免产生气泡，立即插入冰中。
农杆菌感受态制备
版本一1.1农杆菌感受态细胞的制备1.1.1. 取-70 C保存的EHA105于含50用/ml链霉素平板划线，28 C培养。
1.1.2. 挑取单菌落接种于 5ml YM液体培养基中，220rpm 28 C振荡培养12-16 hr。
1.1.3. 取2ml菌液转接于100ml YM 液体培养基中，28 C 220rpm 振荡培养至 OD600=0.5。
1.1.4. 转入无菌离心管，5000rpm 离心5min,去上清液。
1.1.5. 加入10ml预冷的0.1M的CaCI2溶液，轻轻悬浮细胞，冰上放置20min °4C 5000rpm离心5min，去上清。
1.1.6. 加入4ml预冷的含15%甘油的0.1M的CaCl2溶液，轻轻悬浮。
1.1.7. 农杆菌悬浮液分装于无菌Eppendof管中，每管200 M冻存于-70 C。
1.2.双元载体转化农杆菌 EHA105取1⑷左右的质粒 DNA加入到200ml EHA105 感受态细胞中，混匀后，冰浴30min , -70 C放置10min。
再在37 C水浴5min或42 C水浴1min，接着冰浴 2min，加入800mlYM 液体培养基 28 C, 175rpm 摇培3hr后涂在含50旧/ml Kanamycin 的YM平板上。
28 C培养到形成单菌落。
其中 YM 可以用LB代替，第一次的 CaCl2 溶液可用溶液（0.1MCaCI2 0.01Tris-HCI （7.0）0.01 DTT ）替代。
版本二普通农杆菌感受态制备与转化：1. 挑单菌落于2ml YEP培养基（含Rif20mg/ml ）中28 C过夜活化。
2. 取2ml过夜菌液接种于 50ml YEP培养基中28 C生长至 OD600约等于0.5左右（4hr ）。
3. 5k rpm离心5分钟。
4. 在10ml 0.15M NaCI 中悬浮细胞。
5. 5 krpm 离心5分钟，悬浮细胞于 20ml冰预冷的CaCI2。
C43(DE3)感受态细胞使用说明书
C43(DE3)感受态细胞使用说明书OverExpress C43(DE3) Chemically Competent Cell 产品说明书产品规格OverExpress C43(DE3) ：10×100μlpUC19（control vector）：10pg/μl10μl保存条件：-80℃基因型F–ompT hsdSB (rB- mB-) gal dcm (DE3)产品说明OverExpress C43(DE3) 、OverExpress C41(DE3) 两个菌株均起源于 BL21(DE3) ，其优点是可以高效表达毒性蛋白或疏水性蛋白。
OverExpress C41(DE3) 跟BL21(DE3) 的区别在于其基因组含有至少一个未知突变，这个未知突变使其获得了高效表达毒性蛋白（1-5）的能力，此突变位点参与大肠杆菌表达毒性蛋白时的细胞死亡途径；OverExpressC43(DE3) 来源于OverExpress C41(DE3) ，是通过筛选OverExpress C41(DE3)对另一个不同毒性蛋白的抗性菌株获得。
OverExpress C43(DE3) 菌株具有比OverExpress C41(DE3)更强的表达毒性蛋白和疏水性蛋白的能力，所以说OverExpress C43(DE3)菌株与BL21(DE3)相比拥有至少两个未知突变，正是这两个未知突变使其获得了更广泛的表达毒性蛋白或疏水性蛋白的能力。
此菌株含有DE3区，可同时表达T7 RNA聚合酶和大肠杆菌RNA聚合酶，可用于pET系列，pGEX，pMAL等质粒的蛋白表达。
High5TM系列OverExpressC43(DE3) 感受态细胞由特殊工艺制作，经pUC19质粒检测转化效率可达5×108 cfu/μg DNA。
操作方法1. 取100 μl冰上融化的OverExpress C43(DE3)感受态细胞，加入目的DNA并用指尖轻轻拨打管底混匀(避免用枪吸打)，冰上静置25分钟。
AGL-1根癌农杆菌使用说明
菌株以 28℃有氧的培养方式在 LB 或 YEB 上培养，用 30%的甘油可保藏菌
种 。
操作说明：
1，本品包含一份甘油菌，使用本甘油菌时可以不用完全融解，在甘油菌表
面蘸取少量涂板或进行液体培养即可。也可以完全融解后使用，但随着冻融次数
的增加，细菌的活力会逐渐下降。
菌株复壮：
用无菌吸管吸取菌悬液，转移到复溶液滴瓶中。做好标识，在适宜温度下培
养。细菌在 30-‐35℃培养箱中培养 24-‐48h，真菌在 23-‐28℃培养箱中培养 24-‐72h
（必要时，可适当延长培养时间）。
菌 株 传 代 ：
将得到的菌株的新鲜培养物转接到适宜的固体培养基及液体培养基中（尽量
基因（vir 基因是 T-‐DNA 插入植物基因组必需的元件， pTiBo542DT-‐DNA
质粒自身的 T-‐DNA 转移功能被破坏，但可以帮助转入的双元载体 T-‐DNA
顺利转移）。pTiBo542DT-‐DNA 型 Ti 质粒含有筛选标签：strep，赋予 AGL1
菌 株 链 霉 素 抗 性 ，适 用 于 水 稻 、拟 南 芥 、杨 树 等 植 物 的 转 基 因 操 作 。AGL1
AGL-1 根癌农杆菌
编号
名称
北京华越洋生物 NRR01250
AGL-‐1 根癌农杆菌
基 本 信 息 ：
名称：AGL-‐1 根癌农杆菌
规格：300ul 甘油菌
储8 RecA (rif R/carbR) Ti pTiBo542DT-‐DNA (strepR) Succinamopine
2，为保证菌种纯正，避免其它细菌污染，尽量先划平板，然后再挑单克隆
TOP10感受态细胞说明书
TOP10 感受态细胞说明书1.感受态细胞必须用干冰运输，融化后的感受态细胞不能再冻结储存。
如果用户收到感受态细胞后发现泡沫箱中的干冰已经挥发殆尽，应予以拒收。
2.收到感受态细胞后应立即储存在-70℃以下的冰箱中，在6个月内使用不影响转化效率；感受态细胞不可反复冻融，不要与限制性内切酶等经常使用的分子生物学试剂放在一起，避免因温度的波动而影响的效率。
技术支持杭州新景生物试剂开发有限公司研发部：E-mail:technical@，电话：400-0099-857。
产品介绍本公司生产的TOP10感受态细胞是采用大肠杆菌TOP10菌株经特殊工艺处理得到的感受态细胞，可用DNA的化学转化。
经pUC19质粒检测，转化效率可达108cfu/μg。
每支感受态可以酌情分装使用，降低了实验的成本。
质量稳定，使用方便，质优价廉。
TOP10菌株介绍基因型：F- ，mcrAΔ(mrr-hsd RMS-mcrBC)，φ80，lacZΔM15，△lacⅩ74，recA1，ara Δ139Δ(ara-leu)7697，galU/galK ，rps，(Str R) endA1，nupG。
特点：本菌株是一种常用于质粒克隆的菌株。
适用于高效的DNA克隆和质粒扩增，能保证高拷贝质粒的稳定遗传。
其φ80、lacZ△M15基因的产物可与pUC载体编码的β-半乳糖苷酶氨基端实现α互补，可用于蓝白斑筛选。
质量标准1. 使用1 ng pUC19 Plasmid质粒DNA转化100 µl Competent Cells DH5α测试，产生的菌落数＞1×108 transformants/1µg pUC19 Plasmid 。
2. β-半乳糖苷酶、α-互补性的确认：对E.coli Competent Cells TOP10使用pUC19 DNA进行转化后，在含有100 µg/ml的Ampicillin、40µg/ml的X-Gal的L-琼脂平板培养基上，产生蓝色菌落。
感受态细胞说明书
● 细胞浓度
1-2×109 cells/ml
●保 存
-80℃ 注意： -80℃或更低温度下保存。如果保存温度不能恒定，转化降低将会降低。请使用附带的 pUC19 对
照质粒来确认保存细胞的转化效率。不能液氮保存。
● 参考文献
Hanahan, D. (1983) J.Mol.Biol. 166, 557.
X-Gal : 5-Bromo-4-Chloro-3-Indolyl-β-D-Galactoside
● 使用方法
质粒载体转化 1. E.coli DH5α Competent Cells 使用前在冰上融化。 2. 轻微混匀，取 100 μl 装入 14 ml 圆底 tube 中（BD Falcon 352059 或 352057）。
注意：不能剧烈震荡混合细胞。 3. 加入 DNA 样品（建议≤10 ng）。 4. 冰中放置 30 分钟。 5. 42℃放置 45 秒。 6. 冰中放置 1-2 分钟。 7. 添加 SOC 培养基（预先在 37℃保温）至终体积 1 ml。 8. 37℃振荡培养 1 小时（160-225 rpm）。 9. 取适量涂布于选择培养基*。 10. 37℃过夜培养。
● β-半乳糖苷酶的α-互补性
当使用 pUC19 plasmid 进行转化时，含有 100 μg/ml Ampicillin 和 60 μg/ml X-Gal 的 L-agar plate 出 现蓝色菌落。
● Genotype
E.coli DH5α : F−, φ 80dlacZ ΔM15, Δ(lacZYA -argF )U169, deoR , recA1 , endA1 , hsdR17 (rK−, mK+), phoA, supE44 , λ−, thi -1, gyrA96 , relA1
AGL1 感受态细胞
AGL1感受态细胞
AGL1 Competent Cell
北京华越洋
规格
AGL1 ：10×100μl
pCAMBIA2301（control vector）：10ng/ul, 10ul
保存条件：-80℃
华越洋AGL1感受态细胞说明
华越洋生产的AGL1 农杆菌感受态细胞具有利福平抗性，适用于水稻、拟南芥、杨树等植物的转基因操作，经pCAMBIA2301 质粒检测转化效率高达103cfu/μg。
注意事项
1. 感受态细胞最好在冰上融化。
2. 混入质粒时应轻柔操作。
3. 转化高浓度的质粒可相应减少最终用于涂板的菌量。
操作方法
1. 取-80℃保存的农杆菌感受态于冰上或室温融化。
2. 每100μl 感受态加1ug 质粒DNA 混匀，依次于冰上静置10 分钟、液氮5 分钟、37℃5 分钟、冰浴5 分钟。
3. 加入700μl 无抗生素的2YT 或LB 液体培养基，于28℃振荡培养2~3 小时。
4. 5000rpm 离心1 分钟收菌，留取100μl 左右上清轻轻吹打重悬菌块涂布于含相应抗生素的2YT或LB 平板上，倒置放于28℃培养箱培养2-3 天。
OverExpress C43(DE3)化学表达感受态细胞使用说明
OverExpress C43(DE3)化学表达感受态细胞OverExpress C43(DE3) Chemically Competent CellOVEREXPRESS C43(DE3)感受态细胞基因型：F–ompT hsdSB (rB- mB-) gal dcm (DE3)OVEREXPRESS C43(DE3)感受态细胞说明：OverExpress C43(DE3)、OverExpress C41(DE3)两个菌株均起源于BL21(DE3)，其优点是可以高效表达毒性蛋白或疏水性蛋白。
OverExpress C41(DE3)跟BL21(DE3)的区别在于其基因组含有至少一个未知突变，这个未知突变使其获得了高效表达毒性蛋白(1-5)的能力，此突变位点参与大肠杆菌表达毒性蛋白时的细胞死亡途径；OverExpress C43(DE3)来源于OverExpress C41(DE3)，是通过筛选OverExpress C41(DE3)对另一个不同毒性蛋白的抗性菌株获得。
OverExpress C43(DE3)菌株具有比OverExpress C41(DE3)更强的表达毒性蛋白和疏水性蛋白的能力，所以说OverExpress C43(DE3) 菌株与BL21(DE3)相比拥有至少两个未知突变，正是这两个未知突变使其获得了更广泛的表达毒性蛋白或疏水性蛋白的能力。
此菌株含有DE3区，可同时表达T7 RNA聚合酶和大肠杆菌RNA聚合酶，可用于pET系列，pGEX，pMAL等质粒的蛋白表达。
OverExpress C43(DE3)感受态细胞由特殊工艺制作，pUC19质粒检测转化效率可达5×108 cfu/μg DNA。
OVEREXPRESS C43(DE3)感受态细胞操作方法：1. OverExpress C43(DE3)感受态细胞从-80℃拿出，迅速插入冰中，5分钟后待菌块融化，加入目的DNA（质粒或连接产物）并用手拨打EP管底轻轻混匀(避免用枪吸打)，冰中静置25分钟。
农杆菌的培养与感受态制备
农杆菌的培养与感受态制备农杆菌感受态的制备与转化实验室根癌农杆菌感受态的制备：1. 取保存于-70°__菌株在YEB（含125g/ml链霉素或50mg/l利福平）或平板活化，挑取单菌落接种于5mlYEB（含125g/ml链霉素或50mg/l利福平）或YEP(含50mg/l利福平)液体培养基中，28°C、200rpm 培养24-28h。
2. 取2ml菌液接种至50mlYEB（含125g/ml链霉素或50mg/l利福平）或利福平)中，28°C、200rpm培养至OD600≈0.5左右。
3. 菌液冰浴30min后，4°C、8000rpm离心10min收集菌液。
4. 弃上清，用10ml 0.15mol/l Nacl重悬菌体，4°C、8000rpm离心10min收集菌液。
5. 弃上清，用2ml 20m mol/l Cacl2重悬菌体，加入1ml60%无菌甘油(使其终浓度为20%），每管100μl分装，液氮冻存，-70°C保存。
重组质粒电转入农杆菌中：1. 从大肠杆菌中提取重组质粒，将5μl重组质粒加入农杆菌感受态中，至于冰上30min。
2. 将混合物加入电击杯中，选择“Agr”模式（电压2.2kv，时间4.9ms）电击，加入800μl YEB培养基，28°C、200rpm振荡培养3-5h，8000rpm离心30S （实际操作中不离心，吸取菌液50、100、150ul等多做几个梯度，用无菌水稀释至180ul），弃上清，涂布于YEB平板（含农杆菌和质粒所带抗生素，如Kan和利福平）。
3. 28°C恒温箱中倒置培养48h（24h以上）。
4. 从农杆菌中提取质粒，双酶切，琼脂糖凝胶电泳检测；扩目的片段并测序验证。
5. 转化烟草等实验材料。
YEB培养基：1. 0.5%（w/v）蔗糖，0.1%（w/v）细菌用酵母提取物，1%（w/v）细菌用胰蛋白胨，0.05%（w/v）Mg__，调节pH7.0，121°C灭菌15min。
TOP10 感受态细胞说明书
TOP10感受态细胞说明书货号：C1200规格：10×100ul/20×100ul保存：-70℃保存，运输为干冰包装。
自收货之日起液氮保存至少一年，-70℃保存至少6个月。
产品简介：TOP10感受态细胞是采用大肠杆菌TOP10菌株经特殊工艺处理得到的感受态细胞，可用于DNA的化学转化。
使用pUC19质粒检测，转化效率可达108，-70℃保存六个月转化效率不发生改变。
基因型：F_mcrAΔ(mrr-hsd RMS-mcrBC)φ80lacZΔM15△lacⅩ74recA1deoR araD139Δ(ara-leu)7697galU galK rpsL(StrR)endA1nupG特点：一种用于铺制与培养质粒平板和粘粒平板的重级缺陷的抑制型菌株。
其中φ80lacZΔM15基因的产物可与pUC载体编码的β-半乳糖苷酶氨基端实现α-互补，可用于蓝白斑筛选。
该菌株适用于高效的DNA克隆和质粒扩增，能保证高拷贝质粒的稳定遗传。
操作方法：（以下各步骤均为无菌操作）1、将感受态细胞置于冰上融化，以下实验以100ul感受态细胞为例。
2、向感受态细胞悬液中加入需转化的目的DNA，注意目的DNA的体积不要超过感受态细胞悬液体积的十分之一，轻轻旋转离心管以混匀内容物，冰浴放置30分钟。
3、将离心管置于42℃水浴中60-90秒，然后快速转移到冰浴中放置2-3分钟，注意不要摇动离心管。
4、向离心管中加入500ul无菌无抗的SOC或LB培养基，37℃180rpm振荡培养1小时。
目的是使质粒上相关的抗性标记基因表达，使菌体复苏。
5、取适量已转化的感受态细胞涂布含相应抗生素的SOC或LB平板，37℃倒置培养12-16小时。
涂布用量可根据具体实验来调整，如转化的DNA总量较多，可取100ul左右的转化产物涂板；反之，如转化的DNA总量较少，可取200-300ul的转化产物涂板。
过多菌液可以抑制细菌生长。
如果预计的克隆较少，可通过离心后吸除部分培养液，悬浮菌体后将其涂布于一个平板中。
农杆菌感受态的制备和转化
农杆菌感受态的制备和转化版本一1.1农杆菌感受态细胞的制备1.1.1. 取-70℃保存的EHA105于含50μg/ml链霉素平板划线，28℃培养。
1.1.2. 挑取单菌落接种于5ml YM液体培养基中，220rpm 28℃振荡培养12-16 hr。
1.1.3. 取2ml菌液转接于100ml YM液体培养基中，28℃220rpm振荡培养至OD600=0.5。
1.1.4. 转入无菌离心管，5000rpm离心5min,去上清液。
1.1.5. 加入10ml预冷的0.1M的CaCl2溶液，轻轻悬浮细胞，冰上放置20min。
4℃5000rpm 离心5min，去上清。
1.1.6. 加入4ml预冷的含15%甘油的0.1M的CaCl2溶液，轻轻悬浮。
1.1.7. 农杆菌悬浮液分装于无菌Eppendorf管中，每管200μl冻存于-70℃。
1.2. 双元载体转化农杆菌EHA105取1μg左右的质粒DNA加入到200ml EHA105感受态细胞中，混匀后，冰浴30min，-70℃放置10min 。
再在37℃水浴5min或42℃水浴1min，接着冰浴2min，加入800mlYM液体培养基28℃，175rpm摇培3hr后涂在含50μg/ml Kanamycin的YM平板上。
28℃培养到形成单菌落。
其中YM 可以用LB 代替，第一次的CaCl2 溶液可用溶液（0.1MCaCl2 0.01Tris-HCl（7.0）0.01 DTT）替代。
版本二普通农杆菌感受态制备与转化：1. 挑单菌落于2ml YEP培养基（含Rif20mg/ml）中28℃过夜活化。
2. 取2ml过夜菌液接种于50ml YEP培养基中28℃生长至OD600约等于0.5左右（4hr）。
3. 5k rpm离心5分钟。
4. 在10ml 0.15M NaCl中悬浮细胞。
5. 5 krpm离心5分钟，悬浮细胞于20ml冰预冷的CaCl2。
如不是马上使用，可以将之按200ul分裝储存于-80℃待用。
感受态细胞操作步骤
感受态细胞(DH5α，，TOP10)操作步骤第一天1.配制试剂100mM CaCl2：7.35g CaCl2.2H2O /500 ml dd H2O100mM MgCl2：10.15g MgCl2.6H2O /500 ml dd H2O50%（V/V）甘油1L LB 培养基（1L dd H2O+25g LB粉末）75%（V/V）酒精配制试剂（100mM CaCl2100mM MgCl2）：称取相应质量的固体粉末将其转移到事先清洗干净的烧杯里（若有容量瓶，使用容量瓶），用超纯水清洗量筒三次，加少量dd H2O到烧杯溶解固体粉末，将烧杯里溶液加到量筒里，用少量dd H2O清洗量筒三次，将量筒定容到500 ml，将溶液加到试剂瓶里，留待消毒。
配制50%（V/V）甘油：用量筒量取250ml甘油，甘油较粘稠，加时要小心慢加，将量好后的甘油加到试剂瓶里。
用另一个量筒量取250ml dd H2O，用少量水清洗量取甘油的量筒，最终将所有dd H2O加到试剂瓶里，留待消毒。
2. 准备实验用品：1个500mL摇瓶、2个2 L摇瓶（加有锡箔纸）；3个无抗生素的LB平板（100ml dd H2O+3.7g LB粉末，可倒6-7个平板，下午1点左右做平板，所有感受态平板侧面需划两条线）；2个Kana LB平板（100ml LB加入50ul kana）；3ml巴氏吸管；1.5ml 小管（使用没有拆封过的小管，不需灭菌）1ml 枪头；200ul 枪头；4个400ml 离心管（每管实际装350ml液体）。
3. 灭菌锅消毒：100mM CaCl2；100mM MgCl2；50%（V/V）甘油；LB 培养基，dd H2O（若有灭菌水则不需灭）；500 ml 摇瓶；2 L摇瓶；1ml 枪头；200ul 枪头；6个100ml 离心管。
灭完菌后拿到烘箱中60℃烘干，试剂则放到4℃冰箱中。
4. 涂布感受态细胞平板（以DH5α为例）pm 2:30-3:00⑴戴好手套，清理干净操作台，喷洒75%酒精；⑵将三个无抗性的LB平板放在工作台上，在底部标记上DH5α及日期；⑶倒5-8个Beads在第一个平板上，将beads倾到一边，在空白处加10ul灭菌的dd H2O；⑷第一个将LB平板，移液枪、枪头一起拿到-80℃冰箱那的操作台上，从冰箱迅速拿出装有感受态细胞的管子（三中感受态细胞的管子装在一个大袋子里，灰色为DH5α，紫色为TOP10，管子上有标记），用枪头在管子中搅出一小冰块，将冰块加到10ul 水上，盖好平板盖子，将感受态细胞管子迅速放到-80℃冰箱里，整个步骤要迅速小心。
AGL1 电击 电转感受态细胞使用说明
AGL1 电击/电转感受态细胞AGL1 Electroporation Competent CellAGL1电击/电转感受态细胞基因型：C58 RecA (rif r/carb r) Ti pTiBo542DT-DNA SuccinamopineAGL1电击/电转感受态细胞说明：AGL1菌株为C58, RecA型背景，核基因中含有筛选标签——利福平抗性基因rif和羧苄青霉素抗性基因carb，为了便于转化操作，此菌株携带一无自身转运功能的琥珀碱型Ti质粒pTiBo542DT-DNA，此质粒含有vir基因（vir基因是T-DNA插入植物基因组必需的元件，pTiBo542DT-DNA质粒自身的T-DNA转移功能被破坏，但可以帮助转入的双元载体T-DNA 顺利转移）。
AGL1菌株适用于水稻、拟南芥、杨树等植物的转基因操作。
唯地生物开发的AGL1电转感受态特别适用于大质粒的转化：经pCAMBIA2301质粒(size:11633bp)检测转化效率>105 cfu/μg DNA；经pCAMBIA2301-ZH质粒(size:40kd)检测转化效率可达5×103 cfu/μg DNA。
AGL1电击/电转感受态细胞操作方法：1.0.1 cm电击杯和杯盖从储存液中拿出倒置于干净的吸水纸上5分钟，待其沥干水分，正置5分钟，使乙醇充分挥发，待乙醇挥发干净立即插入冰中，压实冰面，电极杯顶离冰面0.5 cm以方便盖上杯盖，冰中静置5分钟充分降温。
2.取-80℃保存的农杆菌感受态插入冰中5分钟，待其融化，加入1-5 μg质粒DNA（质粒体积不大于6ul，最好用试剂盒抽提，双蒸水溶解），用手拨打管底混匀，立即插入冰中，用200 μl枪头将感受态-质粒混合物快速移到电击杯中，盖上杯盖，空管保留待用。
3.启动电转仪，设置参数：C=25 μF，PC=200 ohm，V=2400 V（此参数为Biorad 推荐，使用者也可按所用电转仪推荐的protocol操作），将电击杯快速放入电转槽中，电击完成快速插入冰中，加入700 μl无抗生素的LB并转移到感受态空管中，28℃振荡培养2~3小时。
TG1感受态细胞使用说明书
3.42℃热击45秒，迅速将离心管转移到冰浴中，冰上静置2-3分钟。
4.每个离心管ห้องสมุดไป่ตู้加入450μL无菌的SOC或LB培养基（不含抗生素），混匀后置于37℃
摇床，150 rpm振荡培养45分钟使菌体复苏。
规格及成分
成分
编号
包装
本产品
BTN80701
0.1 mL×10
运输及保存
干冰运输、-80℃保存，有效期半年。
自备试剂
目的DNA、SOC或LB培养基等
使用方法
1.取感受态细胞置于冰浴中。一次转化感受态细胞的建议用量为50-100μL，可以根据
实际情况分装使用。
以下实验以50μL感受态细胞为例。
2.待感受态细胞融化后，向感受态细胞悬液中加入目的DNA（根据实际情况加入适量
TG1感受态细胞使用说明书
产品及特点
本产品是采用大肠杆菌DH5α菌株经特殊工艺处理得到的感受态细胞，可用于DNA的化学转化。使用pUC19质粒检测本产品，转化效率可达108。本产品为dam-，dcm-的菌株，能够排除dam，dcm甲基化的影响。
菌株基因型为：supEhsdΔ5thiΔ(lad-proAB) F+[traD36proAB+lacIQlacZΔM15]
5.根据实验需求，取适量已转化的感受态细胞，加到含相应抗生素的SOC或LB固体
琼脂培养基上，用无菌的涂布棒将细胞均匀涂开，将平板置于37℃直至液体被吸收，
倒置培养，37℃培养12-16小时。
注意：
1.涂布用量可根据具体实验调整。若转化的DNA总量较多，可取少量转化产物涂布平板；若转化的DNA总量较少，可取200-300μL转化产物涂布平板。若预计的克隆数较少，可通过离心（4,000 rpm，2分钟）后吸除部分培养液，悬浮菌体后将其涂布于平板中。
农杆菌感受态细胞的制备
实验一农杆菌感受态细胞的制备［目的及要求］了解和掌握农杆菌感受态细胞制备的原理和方法。
［实验原理］在利用根癌农杆菌介导的基因转化中，首先要获得含有目的基因的农杆菌工程菌株。
在基因工程操作中，感受态细胞的制备和质粒的转化是一项基本技术。
感受态是细菌细胞具有的能够接受外源DNA的一种特殊生理状态。
农杆菌的感受态可用CaCl2处理而诱导产生。
将正在生长的农杆菌细胞加入到低渗的CaCl2溶液中，0℃下处理便会使细菌细胞膜的透性发生改变，此时的细胞呈现出感受态。
制备好的农杆菌感受态细胞迅速冷冻于-70℃可保存相当一段时间而不会对其转化效率有太大的影响。
［实验仪器、材料和试剂］一、仪器：超净工作台，恒温摇床，冷冻高速离心机，高压灭菌锅，冰箱，-70℃超低温冰柜，分光光度计，接种针，10ml试管，50ml离心管，1.5ml 离心管，冰浴，微量进样器及吸头。
以上玻璃仪器和离心管需在用前灭菌，灭菌条件：120℃，15分钟。
二、材料：土壤农杆菌LBA4404菌株或其它农杆菌菌株三、试剂：YEB液体培养基（1L）：酵母提取物1g，牛肉膏5g，蛋白胨5g，蔗糖5g，MgSO4·7H2O 0.5g，pH7.0，高压灭菌。
利福平（Rif）储液：50mg/ml20mM CaCl2，高压灭菌。
［实验步骤］1、挑取根癌农杆菌LBA4404单菌落于3ml的YEB液体培养基（含Rif50mg/l）中，28℃振荡培养过夜；2、取过夜培养菌液1ml接种于50mlYEB（Rif 50mg/l）液体培养基中，28℃振荡培养至OD600为0.5；3、取2ml菌液，13000rpm，离心30sec, 弃上清；4、加入1000µl 20mM CaCl，使农杆菌细胞充分悬浮，冰浴30min；25、13000rpm，离心30sec，弃上清，置于冰上，加入500µl预冷的20mMCaCl，充分悬浮细胞，冰浴中保存，24hr内使用，或液氮中速冻1min，置-70℃2保存备用。
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华越洋⽣生物提供 QQ: 1733351176
A GL1农杆菌感受态细胞使⽤用说明书
编号 名称 产品规格
北京华越洋生物GX9131-‐100s AGL1农杆菌感受态细胞 10*100UL
北京华越洋生物GX9131-‐100 AGL1农杆菌感受态细胞 20*100UL
基本信息：
AGL1农杆菌感受态细胞基因型:
C58 R ecA (rif R/carb R) T i p TiBo542DT-‐DNA (strep R) S uccinamopine
产品说明：
AGL1菌株为C58, RecA型背景，核基因中含有筛选标签——利福平抗性基因rif和羧苄青霉素抗性基因carb，为了便于转化操作，此菌株携带一无自身转运功能的琥珀碱型Ti质粒 pTiBo542DT-‐DNA，此质粒含有vir 基因（vir基因是T-‐DNA插入植物基因组必需的元件， pTiBo542DT-‐DNA 质粒自身的T-‐DNA转移功能被破坏，但可以帮助转入的双元载体T-‐DNA 顺利转移）。
pTiBo542DT-‐DNA型Ti质粒含有筛选标签：strep，赋予AGL1菌株链霉素抗性，适用于水稻、拟南芥、杨树等植物的转基因操作，经pCAMBIA2301质粒检测转化效率可达5×103cfu/μg。
操作⽅方法:
1. 取-‐80℃保存的农杆菌感受态于室温或手心片刻待其部分融化，处于冰水混合状态时插入冰中。
每100μl感受态加1ug（体积不大于10ul）质粒DNA，用手拨打管底混匀，依次于冰上静置5分钟、液氮5分钟、37℃水浴5分钟、冰浴5分钟。
2. 加入700μl无抗生素的LB或YEB液体培养基，于28℃振荡培养2~3小时。
3. 6000rpm离心一分钟收菌，留取100μl左右上清轻轻吹打重悬菌块涂布于含相应抗生素的LB或YEB平板上，倒置放于28℃培养箱培养2-‐3天。
华越洋⽣生物提供
QQ: 1733351176
使⽤用注意事项:
1. 感受态细胞最好在冰上融化。
2. 混入目的DNA 时应轻柔操作。
3. 当平板只含有50ug/ml k an 时，28℃培养48h 即可；平板中同时加入50ug/ml k an ，20ug/ml r if 时，需28℃培养60h ；如果使用的平板含有50ug/ml r if 则需要28℃培养72-‐90h 。
保存条件：
-‐80℃
AGL1农杆菌感受态细胞其他相关产品： DB3.1感受态细胞 EHA105感受态细胞 JM110感受态细胞 XL1-Blue 感受态细胞 XL2-Blue 感受态细胞 NMY51酵母感受态细胞 LBA4404感受态细胞 T1感受态细胞
EGY 酵母感受态细胞 TKB1感受态细胞 AD494感受态细胞 RN4220感受态细胞 Y2HGold 感受态细胞 Rosseta(DE3)感受态细胞 Y1HGold 酵母感受态细胞 TG1感受态细胞 HB101感受态细胞 M15感受态细胞
XL10-Gold 感受态细胞 DH10B 感受态细胞
AGL1农杆菌感受态细胞 Origami 感受态细胞 BLR 感受态细胞 ER2738感受态细胞 MC1061感受态细胞 TOP10感受态细胞
GV3101感受态细胞 EGY48感受态细胞 TB1感受态细胞
Y187酵母感受态细胞 AH109酵母感受态细胞 JM109感受态细胞
stbl3感受态细胞，stbl3 sure 感受态细胞
HMS174感受态细胞 DMT 感受态细胞 B834感受态细胞 EHA103感受态细胞 C41(DE3)感受态细胞。