TOP10 感受态细胞使用说明书
感受态细胞
感受态细胞（competent cell）：理化方法诱导细胞，使其处于最适摄取和容纳外来DNA的生理状态。
、CaCl2法：操作：1.将快速生长的大肠杆菌置于经低温（0℃）预处理的低渗氯化钙溶液中，便会造成细胞膨胀，细胞膜通透性发生变化，极易与外源DNA 相粘附并在细胞表面形成复合物。
2.此时，将该体系转移到42℃下做短暂的热刺激，外源DNA就可能被细胞吸收。
进入细胞的外源DNA分子通过复制、表达，实现遗传信息的转移，使受体细胞出现新的遗传性状。
3.将转化后的细胞在选择性培养基上培养，筛选出带有外源DNA 分子的阳性克隆。
意义：将构建好的载体转入感受态细胞进行表达，不仅可以检验重组载体是否构建成功，最主要的是感受态细胞作为重组载体的宿主可以进行后续实验，如蛋白质表达纯化等工作。
实验五大肠杆菌感受态细胞的制备及转化宁夏大学生命科学学院实验内容：利用化学转化法（CaCl2）制备感受态细胞，并进行转化检测感受态细胞的活性及转化效率。
实验目及原理：掌握大肠杆菌感受态细胞的制备及转化方法细菌处于容易吸收外源DNA的状态叫感受态。
转化是指质粒DNA或以他为载体构建的重组子导入细菌的过程。
其原理是细菌处于0℃，CaCl2低渗溶液中，菌细胞膨胀成球形。
转化混合物中的DNA行程抗DNA酶的羟基-钙磷酸复合物粘附于细胞表面，经42℃短时间热击处理促进细胞吸收DNA复合物。
将细菌防止在非选择性培养基中保温一段时间，促使在转化过程中获得新的表型得到表达，然后将此细菌培养培养物涂在含有氨苄青霉素的选择性培养基上培养观察。
实验仪器：超净工作台；低温离心机；恒温摇床；恒温箱；恒温水浴锅；-70度冰箱；制冰机；分光光度计；微量移液枪等。
实验材料：E. col i DH5α菌株: Rˉ,Mˉ,Ampˉ；pBS质粒DNA；eppendorf管。
试剂1.LB固体和液体培养基2.Amp母液3.含Amp的LB固体培养基:将配好的LB固体培养基高压灭菌后冷却至60℃左右,加入Amp储存液,使终浓度为50ug/ml,摇匀后铺板。
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.。
BL21(DE3)感受态细胞使用说明
BL21(DE3)感受态细胞BL21(DE3)感受态细胞简介：BL21（DE3）感受态细胞是采用大肠杆菌BL21（DE3）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107，-70℃保存几个月转化效率不发生改变。
BL21(DE3)菌株适合表达非毒性蛋白。
该菌株是以T7 RNA 聚合酶为表达系统的外源基因蛋白高效表达的宿主。
T7 噬菌体RNA 聚合酶基因的表达受λ噬菌体DE3区的lacUV5 启动子调控，该区整合在BL21 染色体上。
本产品是大肠杆菌BL21(DE3)菌株经特殊工艺处理得到的感受态细胞，可用于DNA的热击转化。
使用pU19持粒检测，转化效率可达107。
BL21(DE3)感受态细胞产品特点·转化效率可达107。
·可用于非毒性蛋白表达。
·长时间保存于-80 ℃，转化效率不发生改变。
保存条件：-80℃BL21(DE3)感受态细胞基因型F–ompT hsdS(rB– mB–) dcm+ Tet r galλ(DE3) endA Hte [argU proL Cam r] [argU ileY leuW Strep/Spec r]BL21(DE3)感受态细胞说明BL21-CodonPlus(DE3)-RIPL菌株来源于Stratagene公司的BL21-Gold 菌株，缺少Lon蛋白酶和OmpT蛋白酶，从而减少对重组蛋白的降解，补充大肠杆菌缺乏的4种稀有密码子( AGA、AUA、CCC、CUA) 对应的tRNA （argU、ileY、proL、leuW），提高外源基因，尤其是富含AT-或 GC-的真核基因在原核系统中的表达水平。
该菌株染色体整合了λ噬菌体DE3区（DE3区含有T7噬菌体RNA聚合酶），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，同时具有四环素，氯霉素，链霉素，壮观霉素抗性。
TOP10F` 感受态细胞
TOP10F` 感受态细胞TOP10F` Chemically Competent Cell 说明书产品规格TOP10F`：10×100μlpUC19（control vector）：10pg/μl 10μl保存条件：-80℃基因型F´{lacIq Tn10 (TetR)} mcrA ∆(mrr-hsdRMS-mcrBC) Φ80lacZ∆M15 ∆lacX74 recA1araD139 ∆(ara-leu)7697 galU galK rpsL endA1 nupG产品说明TOP10F`菌株来源于TOP10菌株。
将F`{lacIq Tn10 (TetR)}因子转入TOP10菌株，即为TOP10F`。
该F`因子携带lacIq 抑制子，可抑制trc，tac，lac等启动子下游基因的表达，从而可以用于一些表达毒性蛋白质粒的扩繁。
recA1和endA1的突变有利于插入DNA的稳定和高纯度质粒DNA的提取。
可用于构建克隆，蓝白斑筛选（如用于蓝白斑筛选，需在培养基中加入IPTG 诱导β-半乳糖苷酶基因的表达）实验，具有四环素抗性。
High5TM系列TOP10F`感受态细胞经特殊工艺制作，经pUC19质粒检测转化效率>107cfu/μg。
操作方法1. TOP10F`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，冰中静置25分钟。
2.42℃水浴热激45秒，迅速放回冰中并静置2分钟，晃动会降低转化效率。
3. 向离心管中加入700μl不含抗生素的无菌培养基（2YT或LB），混匀后37℃，200 rpm复苏60分钟。
4. 5000rpm离心一分钟收菌，留取100μl左右上清轻轻吹打重悬菌块并涂布到含相应抗生素的2YT或LB培养基上。
5. 将平板倒置放于37℃培养箱12-16h，时间过长会产生假阳性小菌落。
注意事项1. 感受态细胞最好在冰中缓慢融化，插入冰中8分钟内加入目标DNA，不可在冰中放置时间过长，长时间存放会降低转化效率。
感受态细胞的选择与使用
【专题】分子克隆实验专题--（五）感受态细胞的选择与使用作者: 空谷幽风(站内联系TA)发布: 2011-04-16分子克隆实验专题--（五）感受态细胞的选择与使用常用感受态细胞的特性：TOP10，DH5α：蓝白斑筛选，高效克隆，质粒抽提JM110：甲基化酶缺陷型SURE：克隆不稳定插入片段，降低同源重组DH10B：文库构建，长片段质粒的克隆（150kp质粒转化）Stbl2：克隆不稳定插入片段，正向重复，逆转录病毒序列，克隆甲基化基因组序列。
Stbl3：克隆慢病毒表达载体中的正向重复序列，降低逆转录病毒载体中长末端重复序列的非目标同源重组。
ν选择适宜的感受态细胞序列的结构，长度，载体种类以及特殊要求等。
ν选择适宜的转化方式热激转化：常规克隆，片段较短（<10kb)电转化: 建库，基因敲除，长片段（>10kb)感受态细胞使用的注意事项ν连接产物或质粒的量效价：1μg标准质粒转化感受态细胞产生的单克隆的数目，单位为cfu。
化学感受态细胞：106，107，108，109cfu电转感受态细胞：108，109，1010cfu常规的克隆根据连接产物和质粒的量选择适当效价的感受态细胞，特殊转化（如建库）则选择最高效价的感受态细胞，一般选用电转。
ν感受态细胞效价测定标准质粒：pUC18, pUC19,pBR322超螺旋，超纯质粒使用量：<5μl, <1ng设立阴性对照，多涂几板(3块以上）取平均值。
ν化学感受态细胞的使用质粒PCR扩环的产物以及进行体内重组的线性片段不宜选用DH5α高效感受态细胞进行转化。
可能原因：DH5α本身不适合这种操作；高效感受态制备的试剂对于这种转化有抑制作用。
可选用TOP10感受态细胞。
ν电转感受态细胞的使用尽量降低转化产物的离子浓度，防止细胞被击穿。
大量的连接产物提纯后进行转化，或者进行透析后再转化，有些进行体内重组的酶切产物进行提纯后进行转化。
ν氨苄平板的卫星菌落氨苄平板涂板后如果时间太长，就会产生卫星菌落，在单克隆周围生成小的克隆。
TOP10感受态制作方法
TOP 10 感受态细胞制备SOB培养基成分每升成分∙0.5% (w/v) 酵母提取物∙2% (w/v) 胰蛋白胨∙10 mM NaCl∙ 2.5 mM KCl∙20 mM MgSO4∙ 5 g酵母提取物∙20 g胰蛋白胨∙0.584 g NaCl ∙0.186 g KCl∙ 2.4 g MgSO4备注：可以用10 mM MgCl2 and 10 mM MgSO4代替20 mM MgSO4。
高压前调pH至7.5。
放入4℃待用。
CCMB80 buffer 每升成分∙10 mM KAc pH 7.0 (10 ml of a 1M stock/L) 0.98g∙80 mM CaCl2.2H2O (11.8 g/L) 11.8g∙20 mM MnCl2.4H2O (4.0 g/L) 4.0g∙10 mM MgCl2.6H2O (2.0 g/L) 2.0g∙10% 甘油(100 ml/L) 100ml备注：用0.1N HCl调pH至6.4（pH过高会使锰离子变成二氧化锰沉淀）。
无菌过滤后在4℃保存待用。
轻微的深色沉淀不影响其使用效果。
步骤：1.把TOP 10菌种接种至固体LB培养基中37℃过夜培养12 h。
2.挑取大小适当菌落至含10ml LB液体培养基的50 ml玻璃瓶中，置于37 ℃恒温摇床中过夜培养（摇床速度不可过慢，应让菌与空气、营养充分结合，以190左右为宜）。
12 h后观察培养液情况，可适当加长培养时间，但不可过久，因为感受态细菌传代越年轻越好。
3.吸取50μl菌液至于250 ml 配置好的SOB培养基中，至于37℃恒温摇床中培养。
开始时每30 min观察一次，以后每10 min观察一次。
当OD600值为0.3时（轻轻摇荡锥形瓶可见轻微云雾状混浊）立即取出把整个液体置于冰盒的冰面以下冷却待用。
4.将要使用的离心管、buffer在使用前均需置于冰上中降温待用。
无菌操作台、移液枪等在实用前均需用紫外消毒、酒精擦拭干净。
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。
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。
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。
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。
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
Stable化学感受态细胞使用说明
Stable化学感受态细胞Stable Chemically Competent CellStable感受态细胞基因型F' proA+B+ lacI q∆(lacZ)M15 zzf::Tn10 (Tet R) ∆(ara-leu) 7697 araD139 fhuA ∆lacX74 galK16 galE15 e14- Φ80dlacZ∆M15 recA1 relA1 endA1 nupG rpsL (Str R) rph spoT1 ∆(mrr-hsdRMS -mcrBC)Stable感受态细胞说明：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，Stable 感受态细胞经特殊工艺制作，pUC19检测转化效率>5×108cfu/μg DNA。
Stable感受态细胞优点及应用:1.与Stbl3相比，基因组含有endA突变，提高了病毒质粒的产量和纯度。
2.比Stbl3生长速度快，倍增时间是Stbl3的1.5倍。
3.基因组中含有fhuA突变，赋予其对噬菌体T1的抗性。
4.具有较高的转化效率，pUC19检测转化效率>5×108cfu/μg DNA。
5.基因组中含有lacZΔM15，可用于蓝白斑筛选实验。
6.特别适合于不稳定DNA片段的克隆，及逆转录病毒/慢病毒载体的构建和扩繁。
Stable感受态细胞操作方法:1. Stable感受态细胞从-80℃拿出，迅速插入冰中，6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。
DH10B 电击感受态细胞使用说明
DH10B 电击感受态细胞DH10B Electroporation Competent CellDH10B 电击感受态细胞基因型F- mcr A ∆(mr r-hsd RMS-mcr BC) φ80lac Z∆M15 ∆lac X74 rec A1 end A1 ara D139 ∆(ara, leu)7697 gal E15 gal K λ- rps L nup GDH10B 电击感受态细胞说明DH10B菌株来源于MC1061菌株，mcrA、mcrBC及mrr突变使DH10B菌株适合于克隆富含甲基胞嘧啶或甲基腺嘌呤的DNA (无论真核生物还是原核生物的基因组DNA都能被高效的转入DH10B中)。
recA1和endA1的突变有利于插入DNA的稳定和高纯度质粒DNA的提取。
φ80dlacZ∆M15标记的存在使DH10B可用于蓝白斑筛选，rpsL赋予其链霉素抗性。
High5TM系列DH10B感受态细胞适用于大质粒的构建或者各种文库构建。
High5TM系列DH10B感受态细胞经特殊工艺制作，经pUC19质粒检测，转化效率>1010cfu/μg。
注意：DH10B 电击感受态细胞只能用于电击转化而不能用于热激转化。
DH10B 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High5TM 系列DH10B感受态细胞放入冰浴中融化，加入1 μl目的DNA (质粒或连接产物)并用手拨打EP管底轻轻混匀立即插入冰中。
A. 测定转化效率使用1 μl 10 pg/μl的对照质粒pUC19；B. 对于连接产物，请用乙醇沉淀DNA后适量TE缓冲液(10 mM Tris HCl, pH7.5; 1 mM EDTA)重悬，保证DNA浓度不超过100 ng/μl。
TOP10感受态制作方法
TOP 10 感受态细胞制备SOB培养基成分每升成分∙0.5% (w/v) 酵母提取物∙2% (w/v) 胰蛋白胨∙10 mM NaCl∙ 2.5 mM KCl∙20 mM MgSO4∙ 5 g酵母提取物∙20 g胰蛋白胨∙0.584 g NaCl ∙0.186 g KCl∙ 2.4 g MgSO4备注：可以用10 mM MgCl2and 10 mM MgSO4代替20 mM MgSO4。
高压前调pH至7.5。
放入4℃待用。
CCMB80 buffer 每升成分∙10 mM KOAc pH 7.0 (10 ml of a 1M stock/L) 0.98g∙80 mM CaCl2.2H2O (11.8 g/L) 11.8g∙20 mM MnCl2.4H2O (4.0 g/L) 4.0g∙10 mM MgCl2.6H2O (2.0 g/L) 2.0g∙10% 甘油(100 ml/L) 100ml备注：用0.1N HCl调pH至6.4（pH过高会使锰离子变成二氧化锰沉淀）。
无菌过滤后在4℃保存待用。
轻微的深色沉淀不影响其使用效果。
步骤：1.把TOP 10菌种接种至固体LB培养基中37℃过夜培养12 h。
2.挑取大小适当菌落至含10ml LB液体培养基的50 ml玻璃瓶中，置于37 ℃恒温摇床中过夜培养（摇床速度不可过慢，应让菌与空气、营养充分结合，以190左右为宜）。
12 h后观察培养液情况，可适当加长培养时间，但不可过久，因为感受态细菌传代越年轻越好。
3.吸取50μl菌液至于250 ml 配置好的SOB培养基中，至于37℃恒温摇床中培养。
开始时每30 min观察一次，以后每10 min观察一次。
当OD600值为0.3时（轻轻摇荡锥形瓶可见轻微云雾状混浊）立即取出把整个液体置于冰盒的冰面以下冷却待用。
4.将要使用的离心管、buffer在使用前均需置于冰上中降温待用。
无菌操作台、移液枪等在实用前均需用紫外消毒、酒精擦拭干净。
博迈德BC101 TOP10感受态细胞
涂布用量可根据具体实验来调整。转化质粒在 10ng 左右，90mm 平皿涂布 100µl ，55mm 平皿涂布 50µl；连接产物的 转化菌液建议离心后倒掉大部分上清，余 200µl，取 100µl 用于涂布。
6.
保留剩余的菌液于 4℃冰箱中，视平板上菌落生长情况决定去留。
1.
相关试剂及培养基的制备方法 LB 液体培养基：称取 10 g,Tryptone，5 g Yeast Extract 和 10 g NaCl 置于 1 L 烧杯中。加入约 800 ml 的去 离子水，完全溶解后用 2mol/L 的 NaOH 溶液调节 pH 值至 7.0。加去离子水定容至 1 L。分装后，121℃高 压灭菌 20中放置 90 秒，然后快速将管转移到冰浴中，使细胞冷却 2 分钟，该过程不要 摇动离心管。
此步骤也可将离心管置于室温进行，时间不需十分准确，夏季或室温较高时，可放置 5-8 分钟左右；如果室温较低， 可延长时间至 8-15 分钟左右。条件允许建议使用 42 ℃ 热激方法。
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。应注意所用 DNA 体积不要超过感受态细胞 悬液体积的十分之一。以下实验以 100μl 感受态细胞为例。
2.
向感受态细胞悬液中加入目的 DNA ( 100μl 的感受态细胞能够被 1ng 超螺旋质粒 DNA 所饱和），轻轻 旋转离心管以混匀内容物，在冰浴中静置 30 分钟。
BMTOP10 感受态细胞
使 用 说 明 书
目录号：BC101 储存条件：-70 ℃ 保存，避免反复冻融 组成
BMTOP10 pUC19, 0.1ng/ l
BC101-01
10×100l 5 l
BC101-02
GBcompetin 感受态细胞制备及转化 说明书
GBcompetin感受态细胞制备及转化使用说明书Web:Tel**************Fax**************Email:**********************产品描述GBcompetin感受态细胞制备试剂盒是一种快速、高效制备化学态感受态细胞的试剂盒，用此试剂盒制备感受态细胞仅需简单的几个步骤，比常规的氯化钙方法更为快速和高效。
由GBcompetin 溶液制备的感受态细胞可用于即时转化也可长期冷冻保存。
使用pUC19 质粒进行检测，转化效率可达到108 cfu/μg DNA。
GBcompetin感受态细胞制备试剂盒提供了快速而方便的制备方法，适用于几乎所有常用的大肠杆菌菌株。
试剂盒组成GB Transformation Kit产品保存条件100 ml SOB 培养基室温50 ml GB-Wash Buffer 4℃20 ml GBcompetin Buffer 4℃使用说明书GB Transformation Buffer Set产品保存条件100 ml Wash Buffer 4℃40 ml Competent Buffer 4℃使用说明书订购信息保存制备感受态细胞所用试剂可在4 0C保存半年。
使用说明A.感受态细胞的制备1.用接种针挑取大肠杆菌（-80 °C甘油保存菌），在LB平板培养基上划线，置于37 °C恒温培养箱中过夜培养至长出单独菌落。
2.挑取10-12个饱满的单菌落至 5 ml SOC培养基中，置于37 °C摇床中过夜培养。
3.在上述过夜培养的菌液中加入甘油使其浓度达到15%，将上述细胞分装于 1.5 ml离心管中，每管1 ml，分装后保存于-80 °C作为种子菌。
4.在100 ml SOB培养液锥形瓶中加入1 ml种子菌，置于37 °C摇床培养至OD600达到0.2-0.4。
✧此步骤大约需要3小时的培养。
✧确保OD值不要超过此范围，否则将不能保证转化效率。
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TOP10 感受态细胞
Cat. No. JH0102-3
保存：-80℃
组分说明
产品简介
本产品是大肠杆菌 TOP10 菌株经特殊工艺处理得到的感受态细胞，可用于 DNA 的热击转化。
TOP10 是一种常用于质粒克隆的菌株，其 φ80lacZΔM15 基因产物可与载体编码的β-半乳糖苷酶氨基端实现α互补，可用于蓝白斑筛选。
使用 pUC19质粒检测，转化效率可达 108，适用于高效的质粒 DNA 克隆并能保证高拷贝质粒的稳定复制。
注意事项
1.转化所有步骤均在无菌条件下操作。
2.感受态细胞应在-80℃下保存，不可多次冻融和放置时间过长，以免降低感受态细胞的转化效率。
3.包装中有0.1 ng/μl 的pUC19DNA ，供对照试验使用。
操作步骤
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50-100 μl ，可以根据实际情况分装使用。
以下实验以100 μl 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的DNA ，通常100 μl 感受态细胞能够被1 ng 超螺旋质粒DNA 所饱和），用移液器轻轻吹打混匀，冰浴30分钟。
3.42℃热击90秒，迅速将离心管转移到冰浴中，冰上静置2-3分钟。
4.向每个离心管中加入900 μl 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。
5.取100 μl 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，直至干燥，倒置平板，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200-300 μl 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。
2） 新制备的固体培养基不易涂干，可将平板正置于37℃直至液体被吸收后再倒置培养。
3） 涂布剩余的菌液可置于4℃保存，如果次日的转化菌落数过少，可以将剩下的菌液再涂布新培养基进行培养。
Cat. No. JH0102-3 Kit Size 10×100 μl TOP10 感受态细胞
10×100 μl Control DNA pUC19，0.1 ng/μl 10 μl。