Difference between revisions of "Team:JSNU-China/Description"

The experimental operation we mainly used

1.EnoGeneCell Counting Kit-8

Detection principle

◆Cell Counting Kit Acronym CCK8 kit is based WST-8 (chemical name: 2- (2-methoxy-4-nitro-phenyl) -3- (4-nitro-phenyl) -5- (2 , 4-sulfophenyl) -2H- tetrazolium monosodium salt) is widely used in cell proliferation and cytotoxicity of fast, high-sensitivity detection kit.

◆WST-8 belonging to MTT upgrade products, works as follows: In the case of electronic coupling agent is present and can be restored Dehydrogenase mitochondria produce highly water-soluble orange formazan product (formazan). Color depth and proliferation of cells is directly proportional to cell toxicity. OD value was measured using a microplate reader at a wavelength in the 450mM indirectly reflect the number of viable cells.

◆CCK8 method is widely used, such as drug screening, cell proliferation assay, cytotoxicity assays, tumor susceptibility testing and detection of biological activity and other factors.

Cell Viability Assay

1, the cell suspension seeded in 96-well plates (100μL / well). The plates were pre-incubated in an incubator (37 ℃, 5% CO2).

2, was added to each well 10 μL CCK8 solution (be careful not to bubble Kong Zhongsheng, they can affect the reading OD values).

3, the plates were incubated in the incubator for 1-4 hours.

4, then determined by comparing the absorbance at 450nm.

5, if the OD value temporarily, can be added to 10 μL 0.1M HCL solution or 1% w / v SDS solution to each well, and the cover plates stored in the dark at room temperature. Within 24 hours measured absorbance change does not occur.

Cell viability * (%) = [A (dosing) -A (Blank)] / [A (0 dosing) -A (Blank)] × 100

2.Molecular Cloning

3.Western blot

Extract proteins

1.1000r/min centrifuge for 5 minutes to remove PBS.

2.Add lysis buffer and shake it.

3.12000r/min freezing centrifuge for 15minutes

4.Add loading buffer and boil for 10 minutes

5.Put it on the ice.

SDS-PAGE

1. Plates:The sealed box with silica gel placed flat on the glass, and then the concave glass and flat glass overlap, the two glass stand up to make contact with the bottom of the desktop, two glass hand clamped into the electrophoresis tank, then insert Xiecha board to moderate degree, you can glue.

2. Polymer gel:Separating gel and stacking gel preparation: The following table sequentially and proportion solution, configuration of 10% separating gel and 4.8% stacking gel.

3.

	Separating gel	Stacking gel
ddH2O	4.95ml	3.05ml
Arc-Bis(30%)	6ml	0.67ml
Tris-Hcl	3.75ml(PH=8.8)	1.25ml(PH=6.8)
10%SDS	150ul	50ul
10%APS	150ul	50ul
TEMED	15ul	10ul

Following the above table, mix them.After separating gel formulation, add it to the gel, be careful not to produce bubbles. Then add water carefully to make surface covered with water.Put them on room temperature about 30-40min. When absorb the water completely, prepare stacking gel according to the above table. Add stacking gel and insert the comb .Finally,carefully remove the sample.

4.Loading:add sample to the sample tank

5.Electrophoresis

Transfer and block

1.transfer the protein to PVDF membrane.

2.block the membrane with 5% milk 1h.

3.add the first antibody and overnight at 4 degrees temperature.

4.wash membrane 15min 3 times.

5.add second antibody 1h at room temperature.

6.wash membrane 15min 3 times.

Expose

Expose and we can see the result clearly.

4.Cell culture

Cell culture technology refers to the number of cells after a lot of training to become a simple single cell or a little more differentiated cells, which are essential aspects of cloning technology. We can get a lot of cells or metabolites through cell culture. Because biological products are derived from the cells, it can be said cell culture technology is biotechnology core, the most basic technology.

Cell recovery

1.We put cryopreserved tubes in 37 degrees temperature water bath pot and shake it until dissolved.We can complete the thawing about one to two minutes.

2.Put cryopreservation solution and 5 ml of culture into a centrifuge tube and Pipet.

3.1000r/min centrifuge for 5 minutes

4.Add the precipitate to complete medium, pipetting, transferred to a Petri dish.

Cell culture

Add 55ml serum and 6ml Dual anti to the medium.

Collect cell

6.Add Trypsin and stop digestionafter cell culture medium plus curled .

7.Absorb the culture medium to completely blow down cells.

8.Transfer the cells to a centrifuge tube and centrifuge for 5 minutes

9.Discard the supernatant, add wash cells with PBS.

10.1000r/min centrifuge for 5 minutes

11.Discard the supernatant and add frozen -80 degrees to cryopreservate.