Team:HFLS H2Z Hangzhou/sub/protocol

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Team Members

Jianan Li
Qingrui Sun
Yiming Rong
Jiayue Guo
Zhiyuan Lu
Meiqi Yuan
Zhengyao Lin
Caiyi Feng
Shuyun Zhang
Tenghao Huang
Yining Huang
Yanyue Zhu

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Protocols

Chemical Transformation

Materials:

LB broth
LB media
Ice
Selection plates

Methods:

1
Thaw 50 µL competent E. coli cells on ice for 10 minutes
2
Add 5 µl DNA from a ligation reaction mix
3
Carefully flick the tube 4-5 times to mix cells and DNA.
4
Place the mixture on ice for 30 minutes.
5
Heat shock at exactly 42°C for exactly 30 seconds.
6
Immediately place the mixture on ice for 2 minutes.
7
Pipette 950 µL of LB media into the mixture.
8
Incubate in the 37°C shaker for 60 minutes.
9
Spread:
1
Pellet cells at 6000 rpm for 2 minutes;
2
Remove and dispense 750 µL of supernatant;
3
Re-suspend cells by light vortexing;
4
Plate resuspended cells.
10
Incubate overnight at 37°C with plates upside down.

Growing Overnight Cultures

Materials:

LB broth
Antibiotic
12 ml culture tube

Methods:

Overnight cultures were prepared under sterile conditions using a alcohol burner.
1
Add 5 ml liquid LB media into 12 ml culture tubes.
2
Add 5 μl of appropriate antibiotic into the broth.
3
Pick a single colony and inoculate the cultures into the LB broth.
4
Seal the tubes and incubate in the 37°C shaker.

Colony PCR

Materials:

Sterile Water
dNTP (25mM)
DMSO
10X buffer
rTaq enzyme
1 E. coli colony
10 µM forward primer
10 µM reverse primer

Methods:

1
Combine 1.6 µL of dNTP (25mM), 1 µL of DMSO, 0.2 µL of rTaq enzyme, 2 µL of 10X buffer, 0.5 µL of 10 µM forward primer, 0.5 µL of 10 µM reverse primer, and sterile water up to 20 µL.
2
Dip a sterile toothpick to dip in a single colony, and dip it in the mixture to add the cells. Note: Do not add too much cells.
3
Incubate in the thermocycler.

Thermocycling

The PCR machine should be set to run the following steps:
Step Temperature (°C) Time
Initial denaturation 95 10 min
25-35 cycles 95 (denaturation)
57 (annealing)
72 (extension) 		30 seconds
30 seconds
40 seconds
Final extension 72 10 min
Hold 4 Indefinitely

Preparation of LB Broth and Agar

LB Broth

Materials:

Tryptone
Yeast extract
NaCl
Purified Water

Methods:

1
Add 10 g Tryptone, 5 g yeast extract and 10 g NaCl to 1 litre purified water.
2
Stir to thoroughly mix.
3
Autoclave.

LB Agar

Materials:

Tryptone
Yeast extract
NaCl
Agar
Purified Water

Methods:

1
Add 15g agar, 10 g Tryptone, 5 g yeast extract and 10 g NaCl to 1 litre purified water.
2
Stir to thoroughly mix.
3
Autoclave.

DNA Gel Electrophoresis

Materials:

Agarose Powder
TAE buffer
Gel mould
EB dye
Gel Tank
8-10 µL DNA ladder
DNA loading dye

Methods:

1
Prepare 8% w/v solution of agarose powder in 1/10 TAE buffer (e.g. 0.8g agarose powder in 100 mL buffer) using a conical flask.
2
Heat the mixture until agarose is completely dissolved. Do not let the solution boil.
3
Pour the solution into a gel mould.
4
Add 3 µL EB dye to the solution. Make sure there are no bubbles in the solution.
5
Allows the solution to set (approx 15-20 minutes).
6
Transfer the agarose gel to a tank, remove the comb and apply:
8-10 µL of the DNA ladder
DNA samples with the corresponding amount of DNA loading dye.
7
Run the gel for 30-45 minutes at 100V.

Ligation

Materials:

Microcentrifuge tube
Ice
1 µL T4 DNA ligase
1 µL 10X T4 DNA ligase buffer
3 µL vector plasmid
5 µL insert DNA (the ratio of plasmid and DNA should be modified according to the concentration of the DNA)
Sterile water

Methods:

1
Add vector plasmid, insert DNA, T4 DNA ligase and T4 DNA ligase buffer to the microcentrifuge tube on ice (add T4 DNA ligase last).
2
Make reaction up to 10 µL using sterile water.
3
Flick the tube 4-5 times to mix.
4
Incubate at 16°C for at least 30 - 60 min for sticky ends or 1-2 hours for blunt ends.

Restriction Digestion

Materials:

Restriction Enzyme: Choose the enzymes according to target sites. Fast Digestion BamHI and XhoI are used for assembly of pET28a.
Plasmid DNA.
Buffer.
Sterile water.

Methods:

1
Combine 1 µL of restriction enzyme 1, 1 µL of restriction enzyme 2, 2 µL of buffer, 10 µL of plasmid DNA, and sterile water up to 20 µL.
Note: the volume of plasmid DNA can be slightly increased if the concentration is too low. The concentration of plasmid DNA in the mixture should be 0.1-0.4 µg/µL.
2
Incubate digestion reaction at 37°C water bath for 30 minutes.