Team:SJTU-BioX-Shanghai/Protocol

Protocol
PCR

Materials:

Buffer
Mg2+
dNTP
Template
F/R-primer
DNA enzyme

Methods:

  1. 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
  1. 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
Restriction Digestion

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

Ligation

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

DNA Gel Electrophoresis

Materials:

Agarose
TBE Buffer
Gel board
EB
DNA×10 or×6 Loading Buffer
Electrophoresis tank
sample
marker 2000 or 15000

Methods:

  1. 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%)
  2. 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
  3. Wait until the solution cools down to 50℃,add 0.01% EB (eg. 3μin 30ml) or non-poisonous dyes instead
  4. Pour the solution into a gel board and insert the comb
  5. Set the gel at room temperature and wait until it completely solidifies
  6. Remove the comb and transfer the gel to the electrophoresis tank
  7. Add samples into pores with 40% DNA Loading Buffer (eg. 2μLoading Buffer in 5μDNA sample)
  8. Run the gel for 20 min at 100V, 100mA
Transformation

Materials:

LB broth
Ice
Selection plates with corresponding antibiotics.

Methods:

  1. Put 50µL competent E. coli cells on ice for 10 minutes
  2. Add 20 µl DNA from a ligation reaction mix or 10-100 ng plasmid
  3. Incubate the mixture on ice for 30 minutes
  4. Heat shock at exactly 42°C for exactly 45 seconds
  5. Place on ice for 3 minutes
  6. add the mixture into 500 µL LB broth
  7. Incubate at 37°C and 200-250 rpm for one hour
  8. 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
  9. Incubate overnight at 37°C with plates upside down.
Purification and Gel Extraction & Plasmid Extraction

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.

Air Pump Filtration

Materials:

vacuum pump
filter paper
cell culture
mould

Methods:

  1. Assemble the pump and place the filter paper into the funnel correctly
  2. Put the mould onto the filter paper
  3. Switch on the vacuum pump
  4. Add 200μl E.coli culture to the center of the mould
  5. Wait until the liquid drained out completely, put out the filter paper
Characterization

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

Wet Lab
Notebook
Wet Lab
Basic Parts