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 | |
Distilled water | Make up to 15 µL |
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)
Aim: to form a level 2, multigene construct from level 1 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 | |
Bpi1 | 0.5 | |
T4 DNA Ligase | 0.5 | |
Distilled water | Make up to 15 µL |
Step | Temperature (°C) | Time (minutes: seconds) |
---|---|---|
1 | 37 | 0:20 |
2* | 37 | 10:00 |
3* | 16 | 10:00 |
4 | 37 | 10:00 |
5 | 65 | 20:00 |
6 | 16 | ∞ |
*Steps 2-3 cycled x3
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 -80°C 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 42°C.
- 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 37°C.
E. coli Electroporation Transformation
PROTOCOL HERE
Making electrocompetent Agrobacterium tumefaciens
PROTOCOL HERE
Agrobacterium tumefaciens Electroporation Transformation
PROTOCOL HERE
Blue- White Selection
Aim: to select colonies in which the lacZ has dropped out of the acceptor, indicating it has been replaced by the desired construct.
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