Worldshaper-nanjing Protocols

Protocols

Home Experiment Protocols

1. PCR (polymerase chain reaction)

Denaturation: Double-stranded of DNA unwind at high temperature and become single- stranded DNA.
Annealing: Primer is connected on the single-stranded DNA.
Extension: Primer is extended when there is polymerase.

2. The program of PCR

This is the PCR program of Taq Polymerase

This is the PCR program of KOD High-fidelity DNA Polymerase

3.DNA gel electrophoresis experiments

Purpose: verify whether the needed DNA fragments have been amplified successfully or the plasmids have been constructed successfully.

Preparing agarose gel:

Weigh 1.2 g Agarose in 250 mL shake flask and then add 100 mL 1xTAE; heated the solution in the microwave oven until the Agarose is dissolved.
Pipette 5uL Nucleic Acid Dye into 250 mL shake flask and mix thoroughly; Then pour solution into the plastic gel box; Wait for 30 minutes until the gel was solidified.

DNA gel electrophoresis:

Material: 1xTAE, 10 x Buffer, DNA Marker, Agarose gel.
Pour 1xTAE solution into the electrophoresis tank until the agarose gel was submerged.
Put the agarose gel into the tank for electrophoresis.
Pipette 3uL of the solution from PCR and DNA Marker into the gel hole
Open the switch and keep it for 30 minutes.
Put the gel under the electrophoresis gel imaging system to verify the if the plasmids or DNA fragments have been constructed/amplified successfully.

M(marker) is used as the control.

4.DNA restriction enzyme digestion

Purpose: The plasmid was linearized by restriction enzymes digestion for the targeted plasmids construction.

Mixing thoroughly, digestion in 37 ℃ constant-temperature bath for 1 h.

5.DNA ligation reaction

Purpose: Clone the targeted DNA fragment into the correct plasmids for functional gene expression.

Mixing thoroughly, reaction in 16 ℃ constant-temperature bath for 6 h.

6.Escherichia coli transformation experiment

Pipette 10 µL DNA ligation solution into 100 µL E. coli DH5α competent cell. Incubate DH5α on the ice for 30min.
Heat shock for 90s in 42 ℃ constant-temperature bath.
Incubate DH5α on ice for 2min.
Spread LB plates with 100 µg/mL ampicillin and incubation at 37 ℃ for 16 hours.

7.Colony PCR

Purpose: Verify whether the plasmids have been constructed successfully.

Prepare the PCR rection solution:

Use pen to divide a new LB plate into 10 checks on the back side.
Use pipette tips to pick the colonies to the above new LB plate checks. At the same time, the pipette tips was mix with the PCR solution every time.
PCR rection as mentioned above.

8.Plasmid Extraction

Purpose: Increase the number of E. coli and extract plasmid from it.

Pick E. coli containing the correct plasmids from the LB plate to the tube with LB medium and ampicillin.
Put the tube into the shaker (200 r/min, 37 ℃, 16h)
Extract plasmids and use Nanodrop to measure the plasmids concentration.

9.Sequencing

Verify whether the sequences from amplified DNA fragments are corrected.

10.Preparation of Y. lipolytica Competent Cells

Grow yeast cells at 30°C in 10 ml YPD broth until mid-log phase (OD600 of 0.8-1.0). The following steps are accomplished at room temperature.

Pellet the cells at 3000 rpm for 4 minutes and discard the supernatant.
Add 10 ml EZ 1 solution to wash the pellet. Repellet the cells and discard the supernatant.
Add 1 ml EZ 2 solution to resuspend the pellet.

At this point, the competent cells can be used for transformations

11.Yeast Transformation

Mix 50 µl of competent cells with 0.2-1 µg DNA (in less than 5 µl volume); add 500 µl EZ 3 solution and mix thoroughly.
Incubate at 30°C for 45 minutes. Mix vigorously by flicking with finger or vortexing (if appropriate for your DNA) 2-3 times during this incubation.
Spread 50-150 µl of the above transformation mixture on an appropriate plate. It is unnecessary to pellet and wash the cells before spreading. Incubate the plates at 30°C for 2-4 days to allow for growth of transformants.

12.Nutrient deficient medium for yeast transformation

13.Starch medium

This starch medium is intended to test the ability of the yeast to turn starch into biofuels.

14.Make the gel for the verify of the protein.

Put the mixed liquid of yeast into the centrifugal 2500 r/min for 4 minutes and suck up the upper layer of liquid for proteins.
15mL gel

Add the mixture into the container and wait it to solidify. After about half an hour, pour the liquid on the upper level and add the following mixture of reagents.

Put the comb on it to make the holes.
Mix the proteins (10uL) with SDS-PAGE Sample Loading Buffer 2X (10uL) and heat them in 95℃ for two minutes
After the gel solidifies, add the mixture and a marker into the hole on the gel, and put the gel into the machine for one hour.
The dye for the gel (500mL):

Dye the gel in the plastic box.
Decolorizer (1L)

Put gel and decolorizer into the plastic pox and put the box on the shaker for 3~4hours.

15.Lipid extraction and fatty acid analysis

Lipids from an appropriate weighted aliquot of dried cells were extracted using the procedure by Folch et al... A measured quantity of dried cells (roughly 0.5 g) was homogenized in 5 mL of 4 M HCl at room temperature for 30 min, resuspended in a mixture of 10 mL chloroform and 5 mL methanol and vortexed for 30 min.

The lipids were separated by evaporating the chloroform. The extracted total lipids were methylated to generate fatty acid methyl esters (FAMEs) as follows: quantitative lipid extracts (roughly 5 mg) were methylated by adding 1 mL hexane and 100 L methanol, after which the samples were vortexed for 10 min and centrifuged for 3 min at 3000g.

An aliquot comprising 800 L of the hexane layer was then transferred into a clean 1.5 mL microcentrifuge tube for Gas Chromatography-Flame Ionization Detector (GC-FID) analysis. Samples of 1 L were injected into a GC-FID (Agilent 6890 GC), equipped with an Agilent HP-5 column, with detector temperature set to 250 ◦C and N2 flow at 1 mL/min. Fatty acid compositions were determined by comparing peak retention times to those of known standard substances. The amount of fatty acids was quantified by comparing the peak area of the analyte to the peak of the standard substance at known concentrations.

16.Nile red for cell staining

1 mL fresh cell was diluted with PBS to OD600=1, and 1 mL diluent was pipetted into 1.5 ml centrifuge tube.
170 µL of isopropanol and 2 µL of Nile red solution (1 mg/ml in DMSO) were added into the tube.
Incubate for 15 minutes in a dark environment.
The supernatant was removed and 200 µL PBS solution was added into the tube.
The excitation wavelength is between 560-650 nm.

Team:Worldshaper-Nanjing/Protocols