Team:SJTU-BioX-Shanghai/Protocol-cn
Materials:
Buffer
Mg2+
dNTP
Template
F/R-primer
DNA enzyme
Methods:
- Combination of all reagents
| Name of reagent | volume (μl) |
| Buffer | 2 |
| Mg2+ | 2 |
| Template | 20 ng for plasmid or 200 ng for genome |
| F/R-primer | 0.6 for each |
| Enzyme | 0.4 |
| Sterile Water | make reaction up to 20μ in total |
- Set the PCR machine to run the following steps:
| Stage | Temperature (℃) | Time |
| initial denaturation | 94 | 2 min |
| 94 (denaturation) | 15 seconds | |
| 30 cycles | 40-55 (annealing) | 30 seconds |
| 68 (extension) | 30 seconds per bp | |
| Final extension | 68 | 7 min |
| Incubate | 12 | Infinite |
Materials:
Restriction Enzyme: Fisher FastDigest Restriction Enzyme
Green Buffer
DNA samplesr
Sterile Water
Methods:
Note: the components listed below are for double digestion!
| Component | volume (μl) |
| Green Buffer | 2 |
| DNA sample | 600 ng for plasmid or up to 200 ng for PCR production |
| Template | 20 ng for plasmid or 200 ng for genome |
| Restriction Enzyme | 1 for each |
| Sterile Water | make reaction up to 20μ in total |
Set up the reaction following the above table and incubate at 37℃ for 15-30 min and do the gel electrophoresis or purification later
Materials:
T4 DNA Ligase
T4 DNA Ligase Buffer
Vector Plasmid
Insert DNA
Sterile Water
Methods:
| Component | volume or mass |
| T4 DNA Ligase Buffer | 2 μ |
| Vector Plasmid | 50 ng |
| Insert DNA | require molar ratio of 3:1 to 7:1 (insert:vector) |
| T4 DNA Ligase | 0.2 μ |
| Sterile Water | make reaction up to 20μ in total |
Make the reaction and incubate at room temperature for 4-6 hours
Note: for how to calculate volumes of vector and insert DNA, we highly recommend NEBbioCalculator
Materials:
Agarose
TBE Buffer
Gel board
EB
DNA×10 or×6 Loading Buffer
Electrophoresis tank
sample
marker 2000 or 15000
Methods:
- Make a 1.0%-1.5% Agarose gel according to the volume of your gel board (eg. add 0.45g Agarose in 30ml TBE if choose a concentration of 1.5%)
- Heat the container with the mixture until it is complete dissolved. When the solution starts boiling, stop heating, shake the container until materials mix well and heat again until the mixture become clear
- Wait until the solution cools down to 50℃,add 0.01% EB (eg. 3μin 30ml) or non-poisonous dyes instead
- Pour the solution into a gel board and insert the comb
- Set the gel at room temperature and wait until it completely solidifies
- Remove the comb and transfer the gel to the electrophoresis tank
- Add samples into pores with 40% DNA Loading Buffer (eg. 2μLoading Buffer in 5μDNA sample)
- Run the gel for 20 min at 100V, 100mA
Materials:
LB broth
Ice
Selection plates with corresponding antibiotics.
Methods:
- Put 50µL competent E. coli cells on ice for 10 minutes
- Add 20 µl DNA from a ligation reaction mix or 10-100 ng plasmid
- Incubate the mixture on ice for 30 minutes
- Heat shock at exactly 42°C for exactly 45 seconds
- Place on ice for 3 minutes
- add the mixture into 500 µL LB broth
- Incubate at 37°C and 200-250 rpm for one hour
- For ligation reaction DNA: 500 µl of each transformation reaction onto a selection plate. For known plasmid: 100-200 µL of each transformation reaction onto a selection plate
- Incubate overnight at 37°C with plates upside down.
Purification of PCR or restriction products was performed according to the OMEGA Cycle Pure Kit D6492.
Gel Extraction was performed according to the OMEGA Gel Extraction Kit D2500.
Plasmid Extraction was performed according to the BIOMIGA Plasmid Miniprep Kit.
Materials:
vacuum pump
filter paper
cell culture
mould
Methods:
- Assemble the pump and place the filter paper into the funnel correctly
- Put the mould onto the filter paper
- Switch on the vacuum pump
- Add 200μl E.coli culture to the center of the mould
- Wait until the liquid drained out completely, put out the filter paper
Materials:
Replica plate with GFP
Appropriate antibiotic
LB broth
96 well plate
Microplate reader
Methods:
1. Pick appropriate clones from the plate to the 5ml LB broth with antibiotic
2. Grow cells in shacking bed and 37℃ until the OD reaches 0.6
3. Seed cells in the 96 well plat at 200μL per well every two hours, put back the rest solutions and keep growing until the OD becomes stable
4. Set 96 well plate in the microplate reader and read data
