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Molecular Biology Presentation

Molecular Biology Presentation. 生醫 三 Group 13 992334 林思慧 992339 黃珮瑄. LB plate. Negative control. Positive control. Ligation 13-1. Ligation 13-2. Plasmid DNA. 13. M. P. L1. L2. 13 *. P *. L1 *. L2 *. 13. P. L1. L2. 13 *. P *. L1 *. L2 *. 8.0 kb. 3.0 kb. 1.0 kb.

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Molecular Biology Presentation

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  1. Molecular Biology Presentation 生醫三 Group 13 992334 林思慧 992339 黃珮瑄

  2. LB plate Negative control Positive control Ligation 13-1 Ligation 13-2

  3. Plasmid DNA 13 M P L1 L2 13* P* L1* L2* 13 P L1 L2 13* P* L1* L2* 8.0kb 3.0kb 1.0kb 0.5kb M:marker L:ligation P:positive Black:person 1 (思慧) Blue:person 2 (珮瑄) * :treated with restriction enzyme

  4. Discussion • 塗菌的時候沒有塗均勻，colonies沒有分散，挑菌的時候不易找到單一的菌落。 • 塗菌時取的菌液量(300μl)太多，菌長得很密。 • 跑DNA電泳時，因為在loadDNA不小心太快，部分DNA會被沖到well外，使得跑出來的兩組band亮度稍微不同。

  5. Summary • 塗盤 • Negative control知道Ampicillin有效抑制雜菌生長 • Positive control中攜帶抗Amp gene的細菌能夠生長 • Ligation的LB plate上都是攜帶抗Amp gene的DH5α菌株 • DNA電泳 • 經酵素切過後，ligation1&2在1kb的位置都有跑出band，即transformation有成功將帶有DNA片段的vector轉入DH5α菌株中。

  6. SDS-PAGE (CBR) Purification No Purification GST GST-D IPTG GST-D GST-D GST-D GST GST GST-D GST GST M - + - + 75kDa + - + - + - + - 28kDa - : No Purification + : Purification

  7. PVDF membrane (Antibodies) Purification No Purification GST-D GST-D GST-D GST GST GST-D GST GST M 75kDa 28kDa

  8. PVDF membrane (CBR) Purification No Purification GST-D GST-D GST-D GST GST GST-D GST GST M 75kDa 28kDa

  9. Discussion • 在封膠的時候有小泡泡，所以膠有龜裂的現象 • 可能配膠時沒有做好，所以不管是maRker還是蛋白跑的結果中會看到糊掉的maRker或染出直線狀痕跡 • Transfer過程沒有完全趕掉泡泡，阻擋protein與PVDF結合，但是在這裡不影響實驗結果的觀察 • 可能Transfer到PVDF membrane上的蛋白量很少，或者抗體作用不完全，導致免疫染色的結果不佳

  10. Summary • 經過IPTG誘導基因表達並加以純化，western blot結果在~28kDa有大量蛋白的表現。 • 由PVDFmembrane上的CBR檢測結果可知PVDF membrane上是有蛋白成功transferred。利用免疫染色更容易探討單一個蛋白的表現。這個部分實驗可以再次做確認。

  11. References • 慧眾生物科技有限公司-1kb DNA Ladder DL1000 http://www.bioman.com.tw/pr_cont.asp?id=3&myarea=1

  12. Thank for your attEntion!

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