Team:NAU-CHINA/Protocol

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PCR

Methods

Reagent Volume/μL
ddH2O Up to 50
2 × Phanta Max Buffer 25
dNTP Mix(10 mM each) 1
Primer F(10 μM) 2
Primer R(10 μM) 2
Phanta Max Super-Fidelity DNA Polymerase 1
template DNA X

Thermocycling Conditions for a Routine PCR:

Step Temperature (℃) Time Cycles
Initial Denaturation 95 3 min 1
Denaturation 95 15 s 25 - 35
Annealing 56 - 72 15 s 25 - 35
Extension 72 30-60 sec/kb 25 - 35
Final Extension 72 2 - 5 min 1
Hold 16 Indefinitely 1

Gel Extraction

According to the E.Z.N.A. Gel Extraction kit:

The Double Enzyme Digestion System

Components (50 μL) Volume/μL
10 rpmreen Buffer 5
Enzyme I 1
Enzyme II 1
DNA 1 - 2 μg
ddH2O Add to 50 μL

Put the system into a 37 °C water bath for half an hour.

Ligation

Components (10 μL) Volume/μL
T4 DNA Ligase 1
10 × T4 DNA Ligase Buffer 1
Plasmid Skeleton molar ratio of Vector: plasmid is 1:3
Insert Gene molar ratio of Vector: plasmid is 1:3
Sterile Water Up to 10 μL

Overnight at 16 ℃.

Colony PCR

Using 2×Taq master Mix/2×Hieff PCR Master Mix:

Reagent Volume/μL
2× Taq master Mix/2×Hieff PCR Master Mix 5
ddH2O 4
Primer F(10 μM) 0.5
Primer R(10 μM) 0.5
E. coli colony X

Thermocycling conditions for a routine PCR:

Step Temperature (℃) Time Cycles
Initial denaturation 95 3 min 1
Denaturation 95 15 s 25 - 35
Annealing 56 - 72 15 s 25 - 35
Extension 72 30-60 sec/kb 25 - 35
Final extension 72 2-5 min 1
Hold 16 Indefinitely 1

If loading on a gel, don’t need to add loading buffer to the mixture because Taq master Mix/Hieff PCR Master Mix contains loading dye.

Plasmid Extraction

According to the Plasmid Extraction Mini kit:

LB Medium(For 100 mL)

Component Mass/g
Tryptone 1
Yeast Extract 0.5
NaCl 1
Sterile water to 100 ml

Autoclave at 121 °C for 20 min.
(1.5 g agar should be added before autoclaving to make solid LB)

Electro-transformation

Preparation of Bacillus subtilis WB800N competent cells

Medium A (LB + 0.5M sorbitol): Weigh 0.5g sodium chloride, 0.25g yeast extract, 0.5g tryptone, 4.5g sorbitol, and dissolve in 50mL ddH2O.

Medium B (0.5M sorbitol, 0.5M mannitol, 10% glycerol): Weigh 4.56g of mannitol, 4.55g of sorbitol, add 5mL of 100% glycerol, and dilute to 50mL with ddH2O

Electroporation medium (0.5M sorbitol, 0.5M trehalose, 0.5M mannitol, 10% glycerol): Weigh 0.45g sorbitol, 0.95g trehalose, 0.46g mannitol and add 0.5mL of 100% glycerol. Make the volume up to 50mL with ddH2O.

Transformation of recombinant plasmids

Recovery medium (LB + 0.5M sorbitol + 0.38M mannitol): Weigh 0.1g tryptone, 0.9g sorbitol, 0.1g sodium chloride, 0.7g mannitol and 0.05g yeast extract. Dissolve them in 10mL ddH2O.

Western Blot

Running the SDS-PAGE Gel

Transferring the protein from the gel to the membrane

Blocking and antibody staining and development

BCA Assay

BCA reagent: Take 50 parts of BCA reagent A and 1 part of reagent B and mix well.
Standard protein solution: Weigh 0.5g bovine serum albumin, dissolve it in distilled water and dilute to 100mL to make a 5mg/mL solution. Dilute ten times when used.

1.Draw a standard curve: Take a 96-well microtiter plate and add reagents in the following table.

After the above reagents ware added, accurately pipet 20μL of sample solution and add 200μL of BCA reagent to the sample, gently shake, then add the above system to the microplate well, keep it at 37℃ for 30-60min, cool to room temperature, take the blank as a control, and place it on the microplate reader at 562nm. For colorimetry, draw a standard curve with the content of bovine serum albumin as the abscissa and absorbance as the ordinate. Take the blank of the standard curve as the control.

2.Measure the absorbance value of the sample, do three sets of replicates for each sample. Find out the protein content of the sample from the standard curve according to the absorbance value of the sample.