Project Design & Brainstorming

Experimental design and material preparation with the instructor

Phase 1: Experimental attempts, including: >Preparation of reagents for bacterial culture and molecular cloning

1. Preparation of LB Medium
2. Activation of pET28a Strain

>PCR and acquisition of the target gene

1. Extraction of pET28a Plasmid
2. Double Digestion with NdeI/XhoI → Target Gene/pET28a Plasmid
3. Agarose Gel Electrophoresis
4. Gel Extraction of the Digested Bands: pET28a/EC2.4.1.5/EC2.4.1.9

 

> Transformation

1. T4 DNA ligase-mediated ligation of pET28a-2.4.1.5 and pET28a-2.4.1.9
2. Buffer preparation
3. Heat shock transformation

1. Learn from the theory class and prepare for the human practice
2. Periodic group discussions

Objectives of Experiment:

1. Plasmid extraction of pET28a-2.4.1.5/2.4.1.9
2. Heat shock transformation into E. coli BL21(DE3)

Objectives of Experiment:

1. PCR identification of monoclonal/transformed colonies in BL21(DE3)
2. Agarose gel electrophoresis

·Agarose Gel Electrophoresis

Component	Amount
Agarose	0.5 g
1×TAE Buffer	50 mL
Nucleic Acid Stain	3 μL

Objectives of Experiment:

1. Preparation of seed culture fermentation broth
2. SDS-PAGE verification of protein expression

SDS-PAGE Separating Gel Concentrations and Optimal Separation Ranges:

SDS-PAGE Gel Concentration	Optimal Separation Range
6% PAGE gel	70-300 kD
8% PAGE gel	30-200 kD
10% PAGE gel	20-80 kD
12.5% PAGE gel	15-60 kD
15% PAGE gel	10-45 kD

Objectives of Experiment:

1. Protein expression and purification
2. Thin layer chromatography
3. Construction of Digestion Model and Detection of Sucrose Changes and Product Formation in Food Digestion Mixture.

2% Sucrose Treatment		Trehalose Treatment
Number	Sample Amount - Time	Number	Sample Amount - Time
1	1-5	26	1-5
2	1-10	27	1-10
3	1-15	28	1-15
4	1-30	29	1-30
5	1-60	30	1-60
6	5-5	31	5-5
7	5-10	32	5-10
8	5-15	33	5-15
9	5-30	34	5-30
10	5-60	35	5-60
11	10-5	36	10-5
12	10-10	37	10-10
13	10-15	38	10-15
14	10-30	39	10-30
15	10-60	40	10-60
16	15-5	41	15-5
17	15-10	42	15-10
18	15-15	43	15-15
19	15-30	44	15-30
20	15-60	45	15-60
21	20-5	46	20-5
22	20-10	47	20-10
23	20-15	48	20-15
24	20-30	49	20-30
25	20-60	50	20-60