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Made in USA
**Plea note the new heat-shock temperature and duration**
See the transformation protocol for details
Catalog Number
230350
Product Name
SoloPack Gold Supercompetent Cells
SoloPack Gold supercompetent cells, 15 single-tube transformations (50 µl each)
Materials Provided
pUC18 control plasmid (0.1 ng/µl in TE buffer), 10 µl
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Certified By
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Quality Controlled By
Shipped on dry ice.
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Shipping Conditions
Ultracompetent cells must be placed immediately at the bottom of a -80°C freezer directly from the dry ice shipping container. Do Storage Conditions
not store the cells in liquid nitrogen. Ultracompetent cells are nsitive to even small variations in temperature. Transferring tubes
from one freezer to another may result in a loss of efficiency.
≥1 × 109 cfu/µg pUC18 DNA
Guaranteed Efficiency
Transformations are performed both with and without plasmid DNA using 50-µl aliquots of cells and 10 pg of pUC18 control DNA Test Conditions报纸平面广告
普及的意思following the protocol outlined below. Following transformation, 5-µl samples of the culture are plated in duplicate on LB agar
plates with 100 µg/ml ampicillin. The plates are incubated at 37°C overnight and the efficiency is calculated bad on the average
number of colonies per plate.
SoloPack Gold cells are tetracycline and chloramphenicol resistant.
Antibiotic Resistance
Genotype and Background
Tet r D(mcrA)183 D(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F´ proAB lacI q Z D M15 Tn10
(Tet r) Amy Cam r]. (Genes listed signify mutant alleles. Genes on the F´ episome, however, are wild-type unless indicated
otherwi.)
SoloPack Gold supercompetent cells* are packaged in single-reaction aliquots, providing high performance and convenience. The
single-tube transformation format eliminates the need to thaw, aliquot, and refreeze cells and allows for fewer pipetting steps becau
the entire transformation reaction occurs in the tube supplied. SoloPack Gold cells are deficient in all known restrictioncoast guard
systems(McrA-, McrCB-, McrF-, Mrr-, HsdR-), preventing the cleavage of methylated DNA. The strain is endonuclea deficient
datainterface
(endA), greatly improving the quality of miniprep DNA, and recombination deficient (recA), helping to ensure inrt stability. The
Hte phenotype increas the transformation efficiency of ligated and large supercoiled DNA and caus the production of large
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colonies. The lacI q ZΔM15 gene on the F´ episome allows blue-white screening for recombinant plasmids.
Transformation Protocol
1. Preheat NZY+ broth or SOC broth to 42°C for u as the outgrowth medium in step 10.
Note: The optimized protocol us NZY+ broth for maximum transformation efficiency. U of SOC results in slightly
lower efficiencies. See Critical Success Factors and Troubleshooting (rever page) for more information.
2. Thaw two aliquots of SoloPack Gold cells on ice (one tube for each of the experimental and control transformations).
3. When the cells have thawed, swirl the tubes to gently mix the cells.
4. Add 0.01-50 ng of the experimental DNA to one tube of cells (e Quantity and Volume of DNA, on rever page, for
guidelines). Dilute the pUC18 control DNA 1:10 with sterile dH2O, then add 1 µl of the diluted pUC18 DNA to the
other tube of cells.
5. Swirl the tubes gently, then incubate the tubes on ice for 30 minutes.
6. Heat-pul the tubes in a 42°C water bath for 30 conds. The temperature and duration of the heat pul are critical
for maximum efficiency. For consistent results, remove any ice trapped around the outside bottom of the tube.
7. Incubate the tubes on ice for 2 minutes.
8. Add 250 µl of preheated (42°C) NZY+ broth or 250 µl of preheated SOC broth and incubate the tubes at 37°C for
1 hour with shaking at 225-250 rpm.
9. Plate ≤200 µl of the transformation mixture on LB agar plates containing the appropriate antibiotic (and containing
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IPTG and X-gal if color screening is desired). For the pUC18 control transformation, plate 5 µl of the transformation
on LB-ampicillin agar plates.
10. Incubate the plates at 37°C overnight. If performing blue-white color screening, incubate the plates at 37°C for at least
17 hours to allow color development (color can be enhanced by subquent incubation of the plates for 2 hours at 4°C).
11. For the pUC18 control, expect 167 colonies (≥1 × 109 cfu/µg pUC18 DNA).
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Blue-white color screening for recombinant plasmids is available when transforming this host strain (containing the lacI q Z D M15 gene on the F´ episome) with a plasmid that provides α-complementation (e.g. Stratagene pBluescript II). When lacZ expression is induced by IPTG in the prence of the chromogenic substrate X-gal, colonies containing plasmids with inrts will be white, while colonies containing plasmids without inrts will be blue. If an inrt is suspected to be toxic, plate the cells on media without X-gal and IPTG. Color screening will be eliminated, but lower levels of the potentially toxic protein will be expresd in the abnce of IPTG.Blue-White Color Screening U of NZY + broth: For the highest transformation efficiencies (1 × 109 cfu/µg pUC18 DNA), u NZY + broth as the outgrowth medium.Quantity and Volume of DNA: The greatest efficiency is obtained from the transformation of 1 µl of 0.01 ng/µl supercoiled pUC18 DNA per aliquot of cells. A greater number of colonies may be obtained by transforming up to 50 ng DNA, although the resulting efficiency (cfu/µg) may be lower. The volume of the DNA solution added to the reaction may be incread to up to 5 µl, but the transformation efficiency may be reduced.Heat Pul Duration and Temperature: Optimal transformation efficiency is obrved when cells are heat-puld at 42°C for 30 conds in the tubes provided. Efficiency decreas sharply when either the temperature or the d
uration of the heat pul is altered.Plating the Transformation Mixture: If plating <100 µl of cells, pipet the cells into a 200-µl pool of medium and then spread the mixture with a sterile spreader. If plating ≥100 µl, the cells can be spread on the plates directly. Tilt and tap the spreader to remove the last drop of cells. Critical Success Factors and Troubleshooting
SOC Broth (per Liter) NZY + Broth (per Liter)Prepare SOB medium: 10 g of NZ amine (cain hydrolysate) 20.0 g of tryptone 5 g of yeast extract 5.0 g of yeast extract 5 g of NaCl 0.5 g of NaCl Add deionized H 2O to a final volume of 1 liter Add dH 2O to a final volume of 1 liter and then autoclave Adjust to pH 7.5 using NaOH and then autoclave Add 10 ml of filter-sterilized 1 M MgCl 2 and 10 ml of Add the following filer-sterilized supplements prior to u: filter-sterilized 1 M MgSO 4 prior to u 12.5 ml of 1 M MgCl 2 Prepare SOC medium immediately before u: 12.5 ml of 1 M MgSO 498 ml of SOB medium 20 ml of 20% (w/v) gluco 2 ml of filter-sterilized 20% (w/v) gluco LB Agar (per Liter) LB-Ampicillin Agar (per Liter)10 g of NaCl 1 liter of LB agar, autoclaved an
girl friendd cooled to 55°C 10 g of tryptone Add 10 ml of 10 mg/ml filter-sterilized ampicillin 5 g of yeast extract Pour into petri dishes (~25 ml/100-mm plate)20 g of agar Add deionized H 2O to a final volume of 1 liter Adjust pH to 7.0 with 5 N NaOH and then autoclave Pour into petri dishes (~25 ml/100-mm plate) Plates for Blue-White Color Screening Prepare the LB agar and when adding the antibiotic, also add 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) to a final concentration of 80 µg/ml [prepared in dimethylformamide (DMF)] and isopropyl-1-thio-β-D-galactopyranoside (IPTG) to a final concentration of 20 mM (prepared in sterile water). Alternatively, 100 µl of 10 mM IPTG and 100 µl of 2% X-gal may be spread on solidified LB agar plates 30 minutes prior to plating the transformations. (For consistent color development across the plate, pipet the X-gal and the IPTG into a 100-µl pool of SOC medium and then spread the mixture across the plate. Do not mix the IPTG and the X-gal before pipetting them into the pool of SOC medium becau the chemicals may precipitate.)Preparation of Media and Reagents
This warranty limits our liability to replacement of this product. No other warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a
particular purpo, are provided by Agilent. Agilent shall have no liability for any direct, indirect, conquential, or incidental damages arising out of the u, the results of u, or the inability to u this product.Limited Product Warranty *U.S. Patent Nos. 5,512,468 and 5,707,841 and patents pending and equivalent foreign patents.
Endnotes英语四级成绩什么时候公布
For in vitro u only. This certificate is a declaration of analysis at the time of manufacture.
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