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1 Biomolecular Engineering Laboratory Fundamental Lab Work 1. Pipet 2. 度理落數 (CFU) 離 7. DNA 8. DNA 9. 離離 10. 金離 11. (initial rate of absorbance increase) 1. Biological Fuel Cell 2. Bacterial Cellulose Production and Applications 3. Antibiotic Peptides Production by Lactic Acid Bacteria 4. Immobilized Enzymes or Cells as Biocatalyst for Biotransformations 行年利兩兩理行行行

Biological Fuel Cell 2. Bacterial Cellulose Production and Applications 3.

2 牢行量不裸露留糖料理行狀切更理切不精理冷離 1. 料 Merck Index 2. 度 3. 硫 β-mercaptoethanol formaldehyde 量若不理 4. 例 acrylamide 神 ethidium bromide SDS 5. 料不 6. 度切

3 Pipet 量 (micropipet) 行量都了量識 A. 1. Pipetman 不量量 2. 3 量量 (tip) P1000 藍量 P100 量 P10 量量量不塞 4. 量兩 A, 量 B, 量 C, 量量 D, E, 狀

4 B. 1. 量 2. 度量切行 3. 量切不度 70 塞 4. Pipetman Pipetman Pipetman C. 1. 六量離 A~F 2. 列不 L A B C D E F Solution Solution 量離離數 4. 量量 (i) 量留 (ii) 離留

列不 L A B C D E F Solution 1 900 495 999 90 99 5

5 度 A. 度流 Spectra manager 3. Environment Hardware setting Deuterium ( 340nm) 4. Fixed wavelength measurement 5. measurement parameters wavelength OK 6. Autozero 零 B. 1. UV (<340nm ) 2. 若度度 4. 不留 5. 立 6. C. 1. 1mg BSA 10mL BSA 2. BSA 不度 ( mg/ml) 3. 度 OD 度行不度 5. 錄不度度立 BSA 量 R 2 ~0.999 pipet 度

UV (<340nm ) 2. 若 0.8 3. 度度 4. 不留 5. 立 6. C. 1. 1mg BSA 10mL BSA 2. BSA 不度 (0.05 0.

6 A. LB agar plate : 1% (w/v) Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl 5 N NaOH ph % (w/v) Bacto agar 冷 50 B. 1. Kanamycin LB plate (LB+Kan) 2. 落 3. LB+A plate 落連數 (Streak 1) 度 ( Streak 2) 5. (Streak 3) Streak 1 Streak 2 Streak 3 C. LB 1% (w/v) Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl 5 N NaOH ph ml pipet 3 ml LB 2. ( ) 精落落離精 3. 37

LB+A plate 落連數 (Streak 1) 4. 60 度 ( Streak 2) 5. (Streak 3) 6. 37 Streak 1 Streak 2 Streak 3 C.

8 (diauxic growth) When exposed to glucose + lactose, E. coli does not consume lactose until glucose is exhausted, resulting in two exponential growth phases separated by a lag. This is called the diauxie or double growth. Diauxie occurs because synthesis of lactose permease and β-galactosidase is somehow abolished in the presence of glucose. 1. 兩 250 ml 利量 30ml LB 兩 glucose (0.5%) 度 glucose(0.5%) lactose(1%) 2. 1 ml 1 ml 量離 min 1 ml 4. 利度不 OD 600 LB Blank 5. 離行離 6. X- OD 600 兩不

This is called the diauxie or double growth.

9 蘭 A. 理連不若不留若留蘭類兩蘭蘭不 (crystal violet) 藍蘭 (Iodine) 不 CV-I 料 95% 度蘭量留 CV-I 量立不易留蘭量易不易利 CV-I (safranin o) 料蘭蘭留 B Escherichia coli Streptococcus thermophilus 3. 4.

10 來 7. 精行 8. 來精 C. 切度流蘭蘭不不 CV-I 蘭蘭 24 老蘭易留力

11 DNA DNA 立 DNA 狀 DNA 數 kb 行力 DNA DNA DNA 例裂 alkaline lysis boiling method CsCl 裂率廉易理利理 DNA DNA 兩狀復 DNA DNA 亂度易沈來 DNA 兩 DNA 復易離 DNA DNA 離 Solution I Solution II SDS NaOH DNA Solution III DNA 利 DNA 沈 Solution III 離 DNA 留 DNA 精沈 / / 異 [PCI] 留 70 精類量 (mini-prep) 離 DNA CsCl 度度離量 (large-prep) 了量 DNA (molecular cloning) 例行切了 alkaline boiling methods DNA 益不利 (column chromatography) 行量 DNA 更省利裂行 DNA A. DNA ( 裂 ) TE buffer 25mM Tris-HCl, 10mM EDTA, ph8.0 Lysis buffer 0.2N NaOH, 1% SDS Neutralization buffer 5M potassium acetate B. 裂

DNA CsCl 度度離量 (large-prep) 了量 DNA (molecular cloning) 例行切了 alkaline boiling methods DNA 益不利 (column chromatography) 行量 DNA 更省利

12 1. pet30b(+) 3mL (LB+Kan) rpm 離 (6000rpm,1min) L TE buffer L Lysis buffer L Neutralization buffer 離 (12000rpm,5~10min) 量離 L 異 IPA DNA 離 (12000rpm,5~10min) L 70% 精 DNA 離 (12000rpm, 5~10min) 9. DNA 20 L TE buffer DNA agarose 冷切 DNA agarose gel DNA 度 agarose 度了 DNA 不度率不 DNA marker Syber Green Syber Green EtBr 見 C. (agarose, low EEO) 1 TAE % bromophenol blue, 0.25% xylene cyanol, 0.1 M EDTA, 50% glycerol SYBR green stock solution (100X) DNA 量 DNA/HindIII fragments , 9.4, 6.5, 4.3, 2.3, 2.0, 0.56, kb 度 0.5 g/ L λmr

DNA 20 L TE buffer DNA agarose 冷切 DNA agarose gel DNA 度 agarose 度了 DNA 不度率不 DNA marker Syber Green Syber Green EtBr 見 C.

13 D well 1.2% agarose gel 量 agarose 0.3g 1 TAE 25 ml 爐 2. 不 DNA 2 l SYBR Green 2 l loading dye 30 well 行 V 行 bromophenol blue 行 UV transilluminator box 錄 4. 量 DNA 量

14 A. 理力力利略說 1. 不連 (disc-page) 行度 SDS- (SDS-PAGE) 量度 (gradient) 量 SDS 度兩 SDS- 度力 2. 狀立 (16 18 cm) (8 10 cm) 力不狀行行 3. 類 (polyacrylamide) 數 (agarose gel) pi 異 4. 率 ph 8.8 pi 8.8 率量若量狀不率 B. 1. 2, 3. (8x10 cm) Spacer (0.75 mm) Comb (0.75 mm) % acrylanide mix 1.5M Tris(pH8.0) 10% Ammonium persulfate 10% SDS TEMED

15 C. 1. 列不度 SDS-PAGE 2. 易 Isopropanol Isopropanol 4. 3X loading dye 冷 SDS-PAGE 110V 行藍料 6. 兩留切離 ( ) 7. Staining buffer 5 min 50 rpm 不 8. CBR 來藍來 20 ml Destaining buffer 留

16 離離 A. 理離 ion exchange chromatography IEC 離理離不不離不力離度 ph 離來離 5 1. 離 2. 離 3. 行離不易離 4. 離 5. 離離律量離不度降都連離來類 ph ph 離 ph 力來不不 apartic acid glutamic acid ph3~4 glycine alanine arginine lysine ph ph 10~11 B. 1. Buffer A 50 mm Na 2 HPO 4, ph Buffer B 50 mm Na 2 HPO 4, ph 7.0, 0.5 M NaCl 3. DEAE-cellulose C. 1. DEAE cellulose 沈

離離律量離不度降都連離來類 ph ph 離 ph 力來不不 apartic acid glutamic acid ph3~4 glycine

17 降沈降 buffer A 2. 沈降度 0.5 cm buffer A 流流不 3. 濾流 buffer A 流 10 ml 4. bufferb 離流 1 ml 5. 行 SDS-PAGE

18 金離 A. 理若六 His 離 imidazole 離 B. 1. 金 2. Buffer A 50 mm Na 2 HPO 4, ph 8.0; 0.3 M NaCl; 20 mm imidazole 3. Buffer B 50 mm Na 2 HPO 4, ph 8.0; 0.3 M NaCl; 250 mm imidazole C ml 藍塞 buffer A 流 20 ml 塞 2. 弄亂流 3. buffer A 流 buffer A 流 10 ml 4. buffer B 流 1ml 5. 行 SDS-PAGE

Buffer B 50 mm Na 2 HPO 4, ph 8.0; 0.3 M NaCl; 250 mm imidazole C. 1.

19 1. 2. Reagent Stock Final Volume H µL Potassium phosphate buffer, 1M 0.1M 100µL ph8.0 D-alanine 0.3M 0.03M 100µL o-phenylenediamine/o-dianisidine 0.3% 0.03% 100µL Horseraddish peroxidase 0.25U/ L 5U 20µL Enzyme(DAOase) 5µL 3. 度 Time course measurement Measurement parameters 參數列更 Photometric mode=abs Response=quick wavelength=453nm End time=120sec Data Pitch=0.2sec Display=auto 4. 利度力參數來 OD 離金異

高瞻生物教材手冊

高瞻生物教材手冊高瞻生物教材手冊第一單元 : 蛋白質電泳一電泳原理帶電分子在電場中會隨電流而移動, 此現象稱之為泳動, 泳動的大小程度稱之為泳動率 (mobility) 泳動率與分子的電荷密度成正比, 而與分子受到的摩擦力成反比, 摩擦力與分子的