joyce asked in 科學生物學 · 1 decade ago

有請分生高手解釋名詞及翻譯 ~感激不盡!!!

The product of this reaction was then digested with the restriction enzymes EcoRI and PstI. The three vectors chosen to clone EGF were: pET21b(+), pET22b(+) and pET32b(+).When ligating EGF DNA with these three vectors, a HindIII diane was needed, so EGF was first cloned in the pUC118 vector. From this vector it was possible to obtain EGF gene with a simple PCR reaction using the universal primers, FUP (Forward Universal Primer) and RUP (Reverse Universal Primer), and then digesting the product with the restriction enzymes EcoRI and HindIII. With this product and with the three plasmids also digested, a ligation was done. The different transformants obtained were then assessed to see if they were positive for EGF by carrying out digestion with restriction enzymes which would result in fragments of different lengths depending on whether the clone was positive for EGF or not (Fig. 1). Positive clones for each construction were obtained.

Fig. 1. Verification gel of transformants obtained after ligation between EGF gene and the three pET vectors. Samples were loaded on a 1.5% agarose gel. The different lanes are the digestions with ApaI and BglII of pET21b(1) (lanes 1–4), pET22b(+) (lanes 5–8) and pET32b(+) (lanes 9–12). The markers are also shown (1-kb Plus DNA Ladder). Positive clones are marked with an arrow.

需要圖跟我說~在寄你看

Update:

Positive clones 是什麼意思?!

Update 2:

To clone EGF in E. coli, as a plasmid was given to us which contained the EGF gene, pTB361. To obtain EGF gene from pTB361 plasmid a PCR reaction was carried out using specific primers.

Update 3:

謝謝加菲貓...的翻譯..不過我不需要這樣的翻譯~謝謝

Update 4:

謝謝Lionidus回答

在請問Lionidus為什麼要先HindIII diane ?是什麼意思..

Update 5:

請問用限制酶切載體 電泳圖顯現結果如何看

Positive clones 標記跟沒有的lane 差在哪裡?!

2 Answers

Rating
  • 1 decade ago
    Favorite Answer

    Positive clones是指有將target gene 送到載體上,順利連結並穩定表現的

    實在是看不下用翻譯軟體翻譯專業文件的東西了

    你的文章缺了對這一個"product"的描述,導致"The product"用EcoRI and PstI切了之後,跟EGF gene送進pET21b(+), pET22b(+) and pET32b(+).的關係不清不楚

    這也是大家無從幫你的原因

    2008-01-10 01:00:55 補充:

    C loning, expression and purification of human epidermal growth

    factor using different expression systems

    把gene clone到expression vector有相當多的手段

    我對pET的expression vectors不熟也不清楚這vectors的restriction map

    建議你可以去找restriction map後搞清楚為何要用HindIII diane

    EGF DNA大約在4000左右, 因此他挑4000左右的clones為positive clone

    Source(s): , Journal of Chromatography B Volume 788, Issue 1, 5 May 2003, Pages 113-123
  • 1 decade ago

    這是用兩種不同的翻譯軟體,去尋找部分專業名詞,不知你受不受用。

    1.該產品的這種反應,然後消化與限制性內切酶的EcoRI和PstI,當時三個載體選擇克隆表皮生長因子有: pet21b ( + ) , pet22b ( + )和pet32b ( + ) 。這種反應,當時三個載體選擇克隆表皮生長因子結紮時,表皮生長因子的DNA與這三個載體, hindiii戴安需要,使表皮生長因子,是第一只克隆在puc118載體。從這個載體,它是不可能獲得表皮生長因子基因與一種簡單的聚合酶鍊式反應採用通用引物,它有可能fup (著通用引物)和RUP (反向通用引) ,然後在消化產品與限制性內切酶的EcoRI和hindiii 。然後使用這種產品,並與3個質粒,也消化了結紮工作要做。不同的轉化得到的,然後進行評估,以了解他們是否陽性,為表皮生長因子進行消化與限制性內切酶會導致碎片的不同長度取決於是否克隆是積極為表皮生長因子或沒有(圖一) 。陽性克隆,為每個建築獲得了核查凝膠的轉化後,獲得了結紮之間的表皮生長因子基因和三個寵物載體, 樣品被裝上了1.5 %瓊脂糖凝膠。不同車道是digestions與apai和bglii的pet21b ( 1 ) ( 1-4車道) , pet22b ( + ) ( 5-8車道) ,並pet32b ( + ) (車道9-12 ) 。標誌,也顯示( 1 - KB的加DNA梯狀) 。陽性克隆標有一個箭頭。

    2.這反應產品然後消化了與制約酵素EcoRI和PstI。 選上的三傳染媒介克隆EGF是: pET21b (+), pET22b (+)和pET32b (+)。當綁紮EGF脫氧核糖核酸以這三傳染媒介時, HindIII戴安娜是需要的,因此EGF在 pUC118傳染媒介首先被克隆了。 從這傳染媒介使用普遍底漆、FUP ( 向前普遍底漆)是可能的和RUP (反向普遍底漆 ),然後消化產品獲得EGF基因以簡單的PCR反應與制約酵素EcoRI和HindIII。 與這個產品和與也被消化的三質粒,結紮術完成。 然後估計獲得的不同的transformants看他們是否為EGF是正面的由執行的消化與導致不同的長度片段的制約酵素不論克隆為EGF是正面的(。 1). 正面克隆為每建築獲得了。

    。 1. 在結紮術以後獲得的transformants證明膠凝體在EGF 基因和三寵物傳染媒介之間。 樣品在1.5%瓊脂糖膠凝體被裝載了。 不同的車道是消化與pET21b (1) (車道1 V4) , pET22b (+) (車道5 V-8)和pET32b ApaI和BglII (+) (車道9 V12)。 標誌也顯示(1千字節加上脫氧核糖核酸梯子)。 正面克隆標記用箭頭。

    Positive clones陽性克隆

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