PCR

Purpose: to amplify the target gene using beta active.

For gene expressive production
1. Determine two types of cDNA templates, each with two small tubes
2. Mark the centrifuge tube properly; One is wt and the other is active genes
3. Configure a 20 microliter system and calculate the corresponding reagent dosage
CDNA template 2ul
Primers are divided into f and r primers, each with 1 μ l added
DNTPs plus 2ul
TAQ enzyme added 0.5ul
PCR buffer 2ul
Add 11.5ul of ddH2O (double distilled water)
Add according to the volume from large to small, with small volume added to the pipe wall
（All things need to be prepared more in order to eliminate the error）
4. Place 4 PCR systems into a centrifuge for centrifugation (centralized machine requires center of gravity balance), then place them into a PCR instrument for annealing and elongation
94 degrees Celsius for 3 minutes, 55 degrees Celsius for 30 seconds, 72 degrees Celsius for 1 minute
Then repeat 30 times at 94 degrees Celsius for 30 seconds, 55 degrees Celsius for 30 seconds, and 72 degrees Celsius for 1 minute, followed by 10 minutes at 72 degrees Celsius
Last 4 degrees Celsius storage

Western Blot (WB) Overview

Purpose: Identification and quantification of a specific protein in a mixture of proteins extracted from cells or tissues.
1. Sample Collection: In our experiment, we used a cell lysis buffer.
2. Preparation of WB Gel:
Separation Gel (10%):
· Buffer: 625 µL × 4
· DD Water: 820 µL × 5
· 30% Acrylamide: 825 µL × 4
· 10% Ammonium Persulfate: 100 µL
· TEMED: 10 µL
Concentration Gel (5%):
· Buffer: 625 µL × 2
· DD Water: 710 µL × 5
· 30% Acrylamide: 830 µL
· 10% Ammonium Persulfate: 100 µL
· TEMED: 10 µL
Gel Casting:
· Stack the concentration gel on top of the separation gel and insert the comb.
· Smooth the separation gel with a methanol solution.
· Place the small glass plate on top of the large glass plate, and insert it into the electrophoresis chamber, adding running buffer to the chamber.
Sample Loading: Load your samples into the wells.
Gel Running: Run the gel according to your protocol.
Transfer: Sandwich Structure:
· Sponge + Filter Paper + Gel + PVDF Membrane (note to distinguish front and back) + Filter Paper + Sponge
Blocking: Use skim milk as the blocking solution. Prepare a 5% skim milk solution in 1× TBST.
Blocking Procedure:
1. After adding the blocking solution, let it block for 1 hour.
Primary Antibody Incubation:
1. Add the primary antibody and incubate overnight at 4°C.
Secondary Antibody Incubation:
1. Recover the primary antibody, wash with TBST for 15 minutes (repeat three times).
2. Add the corresponding secondary antibody and incubate on a shaker at room temperature for 2 hours, then wash with TBST for 15 minutes (repeat three times).
Detection:
1. Mix developing solution A and B in a 1:1 ratio, then immerse the membrane in the developing solution for 5 seconds before exposing it on the exposure machine.