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Team:NRP-UEA-Norwich/Notebook Protocols
From 2014.igem.org
Lab Protocols
GoldenGate Digestion-Ligation reaction (Level 1)
Aim: to form a level 1 construct (full transcriptional unit) from level 0 modules in a in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.
| Reaction component | Volume(µL) | |
|---|---|---|
| Level 1 acceptor | Ratio 2:1 (Insert: acceptor) | |
| Insert 1 | Ratio 2:1 (Insert: acceptor) | |
| Insert 2 | Ratio 2:1 (Insert: acceptor) | |
| Insert 3 | Ratio 2:1 (Insert: acceptor) | |
| T4 reaction buffer | 1.5 | |
| BSA buffer | 1.5 | |
| BsaI | 0.5 | |
| T4 DNA Ligase | 0.5 |
| Step | Temperature (°C) | Time (minutes: seconds) |
|---|---|---|
| 1 | 37 | 0:20 |
| 2* | 37 | 3:00 |
| 3* | 16 | 4:00 |
| 4 | 50 | 5:00 |
| 5 | 80 | 5:00 |
| 6 | 16 | ∞ |
*Steps 2-3 cycled x26
GoldenGate Digestion-Ligation reaction (Level 2)
PROTOCOL HERE
Making LB Agar
PROTOCOL HERE
Making LB broth
PROTOCOL HERE
E. coli Calcium Chloride Heat Shock Transformation
Aim: to get DNA expression in E. coli.
- Remove chemically competent E. coli cells from the -80oC freezer and thaw on ice.
- Take 1-2 µL of DNA and transfer into a clean 1.5 mL tube.
- Add 50 µL of chemically competent E. coli to the DNA and incubate on ice for 30 mins.
- Preheat water bath to 42oC.
- Heat shock the DNA and E. coli tube for 30-60 sec (not more than 60 sec).
- Transfer back onto ice for 5 mins.
- Add 250-500 µL of LB broth to the tube and incubate at 370C with shaking for 2 hrs.
- Spread plate 100 µL onto plates containing the relevant antibiotics and incubate over night at 37oC.
E. coli Electroporation Transformation
PROTOCOL HERE
Making electrocompetent Agrobacterium tumefaciens
PROTOCOL HERE
Agrobacterium tumefaciens Electroporation Transformation
PROTOCOL HERE
Blue- White Selection
PROTOCOL HERE
Colony PCR
Aim: to determine the size (bp) of the DNA expressed by the colony picked
This helps us to check that the construct is what we expected.DNA mini-prep
Aim
The following protocols were used with QIAGEN QIAprep miniprep kits.- Create an overnight culture- Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with shaking.
- Centrifuge 1-5ml of overnight culture at >8000 for 3 minutes in an Eppendorf tube to form a bacterial pellet; discard the supernatant.
- Re-suspend bacterial pellet in 250µl of P1 Buffer (kept at <5oC).
- Add 250µl of P2 Buffer and invert 4-6 times to mix thoroughly. This reaction is left for no longer than 5 minutes before completing the next step.
- Add 350µl of N3 Buffer and invert 4-6 times to mix thoroughly.
- Centrifuge for 10 minutes at 13,000rpm
- Decant supernatant into a spin column, centrifuge for 30-60 seconds, and discard the flow-through.
- Add 750µl of PE Buffer to the spin column and centrifuge for 30-60 seconds to wash the DNA. Discard the flow-through and centrifuge for a further 1 minute to remove any remaining buffer.
- Remove the top section of the spin column and place in a clean 1.5ml Eppendorf tube. Add 50µl of EB Buffer to the column; let it stand for 1 minute before centrifuging for a further 1 minute to elute the DNA.
Preparation for sequencing
Aim: to sequence our DNA in order to check content of our constructs
- Add 5µl of DNA sample at a concentration of 80-100ng/µl of Plasmid DNA (Diluting with sterile water if Plasmid DNA is at a concentration greater than 100ng/µl) to two separate 1.5 ml Eppendorf tubes.
- Add 5µl of primer 1 (Sense) to the first tube at a total concentration of 5µM (5pmol/µl) and 5µl of primer 2 (AntiSense) to the second tube (at a total concentration of 5µM (5pmol/µl)
- Send off both 10µl samples for sequencing.
Agrobacterium tumefaciens Infiltration
PROTOCOL HERE
Infiltration Analysis
PROTOCOL HERE
Making GoldenGate compatible PSB1C3 'Flipper'
PROTOCOL HERE
Antibiotic Selection
PROTOCOL HERE
