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リソース情報

クロンテック社との、 蛍光タンパク質DsRed2とmCherryの学術利用目的の保存と提供に関するLIMITED USE LICENSE締結

遺伝子発現用・タンパク質産生用クローン&ベクター
  Promoter collection
  Epitope tag fusion clones
  SEREX cDNA クローン
  HLA antigen cDNA clone
  Nakamura & White RFLP clone

遺伝子解析のためのリソース
  培養微生物
  哺乳動物培養細胞
  実験動物個体
  試験管内実験

クローンセット&関連リソース
  Genomic clone
  cDNA clone
  Expression clone
  Libraries
  Others

生物種別リソース
  ヒト、マウス、ラット等哺乳動物
  ツメガエル、ホヤ等その他動物
  酵母・糸状菌
  微生物
  植物

組換えウイルス
  組換えアデノウイルス
  組換えウイルス産生用シャトルベクター

ゲノムDNA
  微生物株由来ゲノムDNA
  マウス系統由来ゲノムDNA

クローン検索
  キーワード検索
  寄託者リスト
  Gene Set Collection

BAC DNA調製


BAC DNA Preparation

  1. Ideally spread BAC on a LB plate to isolate single clolonies. Pick a single colony of BAC into 2ml of 2x LB culture containing a selective antibiotic – incubate at 37 degrees centigrade with shaking for 18-20 hrs.
  2. Collect the culture into a microtube, pellet the cells by centrifuge at 5,000 rpm for 5 mins in a bench top centrifuge, decant the supernatant.
  3. Resuspend the pellet in 300 ul of Qiagen solution P1 (cat no:19051) containing RNAaseA by pipetting – make sure that the cells are completely resuspended and no cell clumps!

    Buffer P1
    50 mM Tris-Cl, pH 8.0; 10 mM EDTA; 100 ug/ml RNaseA

  4. Add 300 ul of Qiagen solution P2 (cat:19052), mix by hand-inversion 4 or 5 times (DO NOT VORTEX), incubate a room temp for 5 mins.

    Buffer P2
    200 mM NaOH; 1% SDS (w/v)

  5. Add 300 ul of Qiagen solution P3 (cat 19053), seal the tubes and mix by inversion – leave on ice for 10 mins.

    Buffer P3
    3 M potassium acetate, pH 5.5

  6. Centrifuge the tubes in a centrifuge at 15 krpm for 15 mins at 4 degrees centigrade.
  7. Transfer the entire supernatant to a fresh tube and precipitate the DNA by adding 700 ul of isopropanol, mix and leave at room temperature for 5 mins.
  8. Centrifuge the tubes in a centrifuge at 15 krpm for 15 mins at 4 degrees centigrade, carefully decant off the supernatant – white pellet should be visible. Wash with 70% EtOH then aspirate off ethanol. You do not need to remove all of the ethanol at this step, but you should minimize it.
  9. Add 50 ul of sterile TE (10mM Tris, pH 8, 1mM EDTA).
  10. Digest 5 ul of the DNA with 10 units of an appropriate restriction enzyme in 20 ul reaction mixture.
  11. Analyze the DNA by a pulsed-field gel electrophoresis.

Related Information

Viability of recombinant E.coli in stab culture

  • Stab cultures of E.coli were stored at different temperatures. Total and only dead cells were stained thiazole orange (TO) and propidium iodide (PI) , respectively. The viability of the E.coli were counted by flow cytometer at indicated days.

Recombineering Information

Related Website

Related Articles

  • Gong, S., Kus, L., Heintz, N. Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis. Nat. Protoc., 5 (10), 1678-1696 (2010). PMID: 20885380
  • Yamaguchi, S., Niwa, R., Kazuki, Y., Ohbayashi, T. Application of a bacterial artificial chromosome modification system for a human artificial chromosome vector. Yonago Acta medica 54:021-031 (2011).