What should this page contain?
- Protocols
- Experiments
- Documentation of the development of your project
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Materials:
LB broth
Ice
Selection plates
Methods:
- Thaw 50µL competent E. coli cells on ice for 10 minutes
- Add:
- 5-10 µl DNA from a ligation reaction mix or
- 10-100ng DNA of a known plasmid
- Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
- Place the mixture on ice for 30 minutes. Do not mix.
- Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
- Place on ice for 5 minutes. Do not mix.
- Pipette 950 µl of room temperature SOC or LB media into the mixture.
- Incubate at 37°C and 200-250 rpm for 60 minutes.
- Mix the cells thoroughly by flicking the tube and inverting.
- Spread:
- For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. For the rest of 900 µL:
- Pellet cells at 8000rpm for 3 minutes
- Remove and dispense 600 µL of supernatant
- Re-suspend cells by light vortexing
- Plate resuspended cells as above
- For known plasmid: 10 & 100 µL of each transformation reaction onto a selection plate. For the rest of 890 µL:
- Pellet cells at 8000rpm for 3 minutes
- Remove and dispense 600 µL of supernatant
- Re-suspend cells by light vortexing
- Plate resuspended cells as above
- For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. For the rest of 900 µL:
- Incubate overnight at 37°C with plates upside down.
Materials:
5 ml LB broth
5 μl antibiotic
Loops
12 ml culture tube
Methods:
Overnight cultures were prepared under sterile conditions using a Bunsen burner
- Add 5 ml liquid LB media into 12 ml culture tubes
- Add 5 μl of appropriate antibiotic into the broth
- Using the loop, pick a single colony and inoculate the cultures by dipping the loop into the LB broth
- Seal the tubes and incubate overnight at 37°C shaking at 200-250 rpm
Materials:
2x Phusion Mastermix
10 µM forward primer
10 µM forward primer
PCR tube
Sterile water
Plasmid DNA
Methods:
For a 25 µL reaction
- In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 1.25 µL of 10 µM forward primer, 1.25 µL of 10 µM reverse primer to a PCR tube on ice, 12.5 µL of 2x Phusion Mastermix, and sterile water up to 25 µL.
Note: It is important to add Phusion Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity - Gently mix the reaction
- If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
- Transfer the PCR tube from ice to a PCR machine to begin thermocycling
For a 50 µL reaction
- In a PCR tube on ice, combine 1-10 ng of plasmid DNA, 2.50 µL of 10 µM forward primer, 2.50 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phusion Mastermix, and sterile water up to 50 µL.
Note: It is important to add Phusion Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity - Gently mix the reaction
- If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly
- Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling
Thermocycling
The PCR machine should be set to run the following steps:
Step | Temperature (°C) | Time |
---|---|---|
Initial denaturation | 98 | 30 seconds |
25-35 cycles | 98 (denaturation) 45-72 (annealing) see Note 1 72 (extension) |
5-10 seconds 10-30 seconds 15-30 seconds per kb |
Final extension | 72 | 2-5 minutes |
Hold | 4 | Indefinitely |
Note 1: Use the NEB Tm calculator should be used to determine the annealing temperature when using Phusion: http://tmcalculator.neb.com/#!/
Materials:
Sterile Water
25 µL RedTaq mastermix
1 E. coli colony
2.5 µL of 10 µM forward primer
2.5 µL of 10 µM reverse primer
Methods:
- Add a single colony of cells to 50 µL of water. Incubate at 95C for a minute to lyse the cells.
- Combine 1 µL cell lysate, 25 µL RedTaq mastermix, 2.5 µL of 10 µM forward primer, 2.5 µL of 10 µM reverse primer, and sterile water up to 50 µL.
Note: It is important to add RedTaq Master Mix last in order to prevent primer degradation caused by the 3 ́→ 5 ́ exonuclease activity - Incubate in the thermocycler - Taq has a lower optimum temperature than Phusion.
Thermocycling
The PCR machine should be set to run the following steps:
Step | Temperature (°C) | Time |
---|---|---|
Initial denaturation | 98 | 30 seconds |
25-35 cycles | 98 (denaturation) 45-72 (annealing) see Note 1 68 (extension) |
5-10 seconds 10-30 seconds 15-30 seconds per kb |
Final extension | 72 | 5-10 minutes |
Hold | 4 | Indefinitely |
Note: If loading on a gel, the RedTaq mix contains loading dye, so don’t add anything else.
Materials:
25 g LB broth (powder)
1 Litre Purified Water
Methods:
- Add 25g LB broth to 1 litre purified water
- Autoclave
Materials:
37 g LB Agar (powder)
1 Litre Purified Water
Methods:
- Add 37g LB Agar to 1 litre purified water
- Autoclave
Materials:
500µl glycerol (80%)
500µl overnight culture in LB
Methods:
- Add 500µl glycerol (80%) to 1.5ml eppendorf tube
- Add 500µl overnight culture in LB
- Store at -80°C
Materials:
Agarose Powder
TAE buffer
Gel mould
5-10 µL SybrSafe
Gel Tank
8-10 µL DNA ladder
DNA loading dye
Methods:
- 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
- Heat the mixture until agarose is completely dissolved. Do not let the solution boil.
- Pour the solution into a gel mould
- Add 5-10 µL SybrSafe® to the solution. Make sure there are no bubbles in the solution.
- Allows the solution to set (approx 15-20 minutes)
- 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 (6X)
- Run the gel for 45-60 minutes at 100V
Materials:
Microcentrifuge tube
Ice
1 µL T4 DNA Lligase
2 µL 10X T4 DNA ligase buffer
50ng Vector Plasmid
Insert DNA
Sterile water
Methods:
- Calculate volumes of vector and insert DNA required using NEBioCalculator ( http://nebiocalculator.neb.com/#!/ligation - required molar ratio of 1:3::vector:insert)
- Add vector plasmid, insert DNA, T4 DNA ligase and T4 DNA ligase buffer to the microcentrifuge tube on ice (add T4 DNA ligase last)
- Make reaction up to 20 µL using sterile water
- Incubate at room temperature for 30 - 60 min for sticky ends or 1-2 hours for blunt ends
Materials:
Restriction Enzyme:
https://www.neb.com/tools-and-resources/interactive-tools/enzyme-finder?searchType=7&recognitionSite=&matchType=1used to see which restriction enzymes are required
10X buffer:https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finderused to see which buffers are required for the particular restriction enzymes
Plasmid DNA
Sterile water
Methods:
- Set up the following reaction, depending on whether test digest or assembly digest is being performed:
Component Test digest
Double digestion (20 µL, for construct analysis)Assemble digest
Double digestion (20-50 µL, for gene assembly)Sterile water to 20 µL to 50 µL 10X buffer 2 µL 2-5 µL Plasmid DNA ~200 ng for test digest, (DNA mass is variable dependent on insert size. Smallest digestion fragment mass should be > 50ng) 1,000 -2,000 ng Restriction enzyme 0.5 µL + 0.5 µL 1 µL + 1 µL - 30 min for Test digest
- 2-3 hours or overnight for Assembly digest