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

Sponsors

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.

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