Team:Mingdao/Notebook
Timeline
Protocol
Bacterial Culture
For plasmid extraction, we normally cultured E. coli carrying plasmids in 6 ml LB broth supplemented with chloramphenicol (34 μg/ml) or ampicillin (100 μg/ml) at 37°C overnight
Plasmid Extraction Procedure
In all of our experiments, we used a mini-prep kit (Presto™ Mini Plasmid Kit) developed by GENEAID BIOTECH LTD., Taiwan. And we followed the manufacture’s instruction for plasmid extraction. Below is the brief protocol provided by Geneaid Biotech Ltd., Taiwan. And you can also go to their website for more information and complete protocol.
PCR for Gene Cloning
We always used a high fidelity DNA polymerase named KOD -Plus- developed by TOYOBO CO., LTD. We followed the manufacture’s instruction. Brief conditions was showed below.
DNA Insertion Check by Colony PCR
We used a kit of Taq polymerase (JMR Holdings) and followed manufacture’s instruction. Brief conditions was showed below.
DNA Gel Electrophoresis
We prepared 1% DNA agarose gels and run gel with 6X FluoroDye DNA Loading Dye (Green) and FluoroBand 1KB (0.25-10kb) Fluoescent DNA Ladder. The images were pictured with Omega Lum™ G Imaging System.
PCR Cleanup & Gel Elution
We used GenepHlow™ Gel/PCR Kit developed by GENEAID BIOTECH LTD., Taiwan and followed the manufacture’s instruction.
Restriction Enzymes
EcoRI, XbaI, SpeI, PstI, MfeI, NotI and NsiI were used in this study and purchased from New England Biolabs (NEB). We have three conditions as described below used for plasmid check and gene cloning, respectively.
Plasmid Check
Gene Cloning (for vectors)
Gene Cloning (for inserts)
Competent Cells
We used and purchased Elite Competent Cells (DH5α) from GENEAID BIOTECH LTD. We followed the provided protocol for transformation procedure and briefly described as below.
Transformation
- Thaw one tube of competent cells on ice from -80°C refrigerator
- Add 5 μl of DNA into 50 ul of competent cells. Operate in a laminar flow and manipulate DNA and competent cells on ice.
- Vortex for 1 sec and stay on ice for 5 min
- Heat shock the cells at 42°C for 1 min
- Incubate on ice for 5 min
- Recover with 500 μl of LB media and shaking in a incubator at 37°C for 1 hr. Operate in the laminar flow.
- Spread the cells onto a LB agar plate supplemented with 34 μg/ml of chloramphenicol or 100 μg/ml of ampicillin. Operate in the laminar flow.
Glucose Assay
The bacteria were culture in LB broth supplemented with 34μg/ml of chloramphenicol at 37°C overnight. The next day, the bacterial culture was adjusted to OD600 = 3 and exchanged with M9 minimal media with 20mM of glucose for 4 hours or at different time points.
Glucose concentration was analyzed with Glucose (HK) Assay Kit (Sigma-Aldrich) according to the manufacturer’s instruction. Briefly, glucose was phosphorylated (G6P) by hexokinase. Then G6P was further catalyzed by G6PDH and the reduced NAHD was formed from the oxidation of NAD, resulting in increasing in absorbance at 340 nm.
Glucose Response Assay
The bacteria carrying the indicated vector were cultured in LB media supplemented with 34 μg/ml of chloramphenicol (Cm) at 37°C overnight. The next day, OD600 was measured and adjusted to 2.5 in M9 minimal media with various concentrations of glucose. The bacteria then were incubated for 4 hours at 37°C. 100 μl of the bacterial culture was put into one well of a black-walled, clear-bottom 96-well microplates (Thermo Fisher Scientific Inc.). The fluorescence intensity was measured using BioTek Synergy H1 Hybrid Multi-Mode Reader System at Ex/Em = 584nm/607nm for RFP or Ex/Em = 488nm/518nm for GFP. The values of OD600 were also measured for bacterial growth. For suicide assay, the culture media were further taken out and diluted 106 times following by spreading onto LB Cm agar plate at 37°C overnight. The third day, the numbers of colonies were counted and bacterial viability was calculated.
