MAX Efficiency™ DH10Bac Competent Cells

Citations & References (4)

Gibco™

MAX Efficiency™ DH10Bac Competent Cells

MAX Efficiency DH10Bacコンピテントセルは、Bac-to-Bac Baculovirus Expression Systemで使用される組換えバクミドの製造に使用されます。DH10Bac E. coli株には詳細を見る

製品番号（カタログ番号）	数量
10361012	5 x 100 μL

製品番号（カタログ番号） 10361012

価格（JPY）

72,300

お問い合わせください ›

5 x 100 μL

MAX Efficiency DH10Bacコンピテントセルは、Bac-to-Bac Baculovirus Expression Systemで使用される組換えバクミドの製造に使用されます。DH10Bac E. coli株には、目的遺伝子のクローンを含む発現バクミドを作成するために、ドナープラスミドpFastBacと再結合できるバキュロウイルスシャトルベクター（バクミド）が含まれています。MAX Efficiency DH10Bacコンピテントセルの特徴は以下のとおりです。

• 一貫した >1 ×10⁸ の形質転換体 /µg の収量
• 少量の希釈されていないライゲーション反応の耐性
• 効率的な青/白コロニースクリーニングのサポート（Φ80lacZΔ M15マーカー）

最大効率DH10Bacコンピテントセルの使用
最大効率DH10Bacコンピテントセルは、目的遺伝子のクローニングを含む組換えpFastBacベクターの宿主となります。DH10Bac細胞は、バキュロウイルスシャトルベクター（bMON14272）とヘルパープラスミド（pMON7142）を有し、pFastBacとbMON14272の部位特異的な組換えをサポートしており、これらの結合を増幅して精製し、昆虫細胞のトランスフェクションおよびそれに続く遺伝子の発現に使用できる高分子量のバクミドを生成します。バクミドの選択および維持に対するカナマイシン耐性は、bMON14272によりもたらされ、テトラサイクリン耐性はpMON7124によりもたらされます。

遺伝子型：F^-mcrA Δ（mrr-hsdRMS-mcrBC） Φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ（ara、leu） 7697 galU galK λ^-rpsL nupG/pMON14272/pMON7124

研究用にのみ使用できます。診断用には使用いただけません。

菌株または酵母株DH10Bac

青／白スクリーニング可

細胞タイプケミカルコンピテント

メチル化DNAのクローニング不可

不安定DNAのクローニング不安定な DNA のクローニングには適していません

F'エピソームを含むF’エピソームが欠落しています

効率> 1x10^8

高スループット適合性ハイスループット非対応（手動）

プラスミドの品質を向上不可

非メチル化DNAの調製非メチル化DNAの調製には適していません

製品ラインDH10Bac、最大効率

製品タイプコンピテントセル

増殖ccdBベクターccdBベクターの増殖には使用できません

数量5 x 100 μL

組換えを抑制不可

出荷条件ドライアイス

T1ファージ－耐性（tonA）不可

形質転換効率レベル中効率 (10^8-10^9 cfu⁄µg)

フォーマットチューブ

種E. coli

Unit SizeEach

組成および保存条件

内容：
• MAX Efficiency DH10Bacケミカルコンピテントセル：5バイアル、各100 µl（合計500 µl）
• pUC19 DNA（0.01 µg/ml）：1 バイアル、100 µl
• SOC 培地：

2 ボトル、各 6 ml、コンピテントセルは-80℃で保存します。pUC19 DNAは-20℃で保存してください。SOC培地は4℃または室温で保存してください。

よくあるご質問（FAQ）

I cannot detect any recombinant fusion protein after using the BaculoDirect Expression Kit. What could be the cause for this and what do you suggest I try?

Please check the construction of your entry clone, and ensure that the insert is in frame with the vector. Analyze the recombinant viral DNA by PCR to confirm the correct size and orientation of your insert after the LR reaction. Sequence your PCR product to verify the proper reading frame for expression of the epitope tag.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting a low-titer P1 viral stock and would like to generate a high-titer stock. What should I do?

To get a high-titer stock, reinfect cells with the P1 stock and generate a P2 high-titer stock. Follow the directions in the BaculoDirect manual on page 18 to generate your P2 stock.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I performed an LR reaction, followed by transfection into Sf9/Sf21 insect cells, but do not see any signs of infection even though it's been 72 hours. What should i do?

Please see our recommendations below:

- Check the LR reaction by PCR analysis prior to transfection into insect cells.
- We recommend using Grace's Insect Cell Culture Medium, Unsupplemented during the transfection experiment instead of serum-free medium, as components in serum-free medium may interfere with transfection.
- Ensure that FBS, supplements, or antibiotics are not included during transfection, as the proteins in these materials can interfere with the Cellfectin II Reagent.
- Use the LR recombination reaction using the pENTR/CAT plasmid as a positive control and Cellfectin II Reagent only (mock transfection) as a negative control.
- Ensure that cells are in the log phase of growth with >95% viability, and the amount of cells are in accordance with the suggestions in the manual.
- Cells may not show signs of viral infection for up to a week depending on transfection efficiency; continue culturing and monitor cells daily for signs of infection.

I see a precipitate in my ganciclovir solution. What can I do?

Warm the ganciclovir solution in a water bath at 37 degrees C for 5-10 min, then vortex for a few minutes. The precipitate should go back into solution.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I am purifying my secreted protein expressed in insect cells with a His-tagged purification column and getting no yield of my protein. Why and what can I do?

Media used to culture insect cells usually have an acidic pH (6.0-6.5) or contain electron-donating groups that can prevent binding of the 6xHis-tagged protein to Ni-NTA. Amino acids such as glutamine, glycine, or histidine are present at significantly higher concentrations in media for growing insect cells than in media for growing mammalian cells, and compete with the 6xHis-tagged protein for binding sites on Ni-NTA matrices. Grace's medium (Thermo Fisher Scientific), for example, contains approximately 10 mM glutamine, 10 mM glycine, and 15 mM histidine.

Dialysis of the medium against a buffer with the appropriate composition and pH (8.0) similar to the lysis buffer recommended for purification under native conditions usually restores optimal binding conditions. Note that depending on the medium used, a white precipitate (probably made up of insoluble salts) can occur, but normally the 6xHis-tagged protein remains in solution. This can be tested by either protein quantitation if using a protein-free medium or by monitoring the amount of 6xHis-tagged protein by western-blot analysis. After centrifugation, 6xHis-tagged protein can be directly purified from the cleared supernatant.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

View all MAX Efficiency™ DH10Bac Competent Cells FAQs

引用および参考文献 (4)

引用および参考文献

Abstract

Molecular characterization of hCTR1, the human copper uptake protein.

Authors: Eisses John F; Kaplan Jack H;

Journal:J Biol Chem

PubMed ID:12034741

'We have expressed hCTR1, the human copper transporter, in Sf9 cells using a baculovirus-mediated expression system, and we observed greatly enhanced copper uptake. Western blots showed that the protein is delivered to the plasma membrane, where it mediates saturable copper uptake with a K(m) of approximately 3.5 microm. We also ... More

Characterization of a heparan sulfate 3-O-sulfotransferase-5, an enzyme synthesizing a tetrasulfated disaccharide.

Authors:Mochizuki H, Yoshida K, Gotoh M, Sugioka S, Kikuchi N, Kwon YD, Tawada A, Maeyama K, Inaba N, Hiruma T, Kimata K, Narimatsu H,

Journal:J Biol Chem

PubMed ID:12740361

Heparan sulfate d-glucosaminyl 3-O-sulfotransferases (3-OSTs) catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to position 3 of the glucosamine residue of heparan sulfate and heparin. A sixth member of the human 3-OST family, named 3-OST-5, was recently reported (Xia, G., Chen, J., Tiwari, V., Ju, W., Li, J.-P., Malmstrom, ... More

Gene characterized for membrane desaturase that produces (E)-11 isomers of mono- and diunsaturated fatty acids.

Authors: Liu Weitian; Jiao Hongmei; Murray Nancy C; O'Connor Marion; Roelofs Wendell L;

Journal:Proc Natl Acad Sci U S A

PubMed ID:11805319

Moth species have evolved integral membrane desaturases that exhibit a wide diversity in substrate specificity, as well as in regiospecificity and stereospecificity of the unsaturated products. We report here the cloning and expression of a single desaturase from the sex pheromone gland of the light brown apple moth, Epiphyas postvittana, ... More

Reconstitution of ATP-dependent leukotriene C4 transport by Co-expression of both half-molecules of human multidrug resistance protein in insect cells.

Authors: Gao M; Loe D W; Grant C E; Cole S P; Deeley R G;

Journal:J Biol Chem

PubMed ID:8910374

Multidrug resistance protein (MRP) confers a multidrug resistance phenotype similar to that associated with overexpression of P-glycoprotein. Unlike P-glycoprotein, MRP has also been shown to be a primary active ATP-dependent transporter of conjugated organic anions. The mechanism(s) by which MRP transports these compounds and increases resistance to natural product drugs ... More