Team:NRP-UEA-Norwich/Notebook Protocols
From 2014.igem.org
Lab Protocols
GoldenGate Digestion-Ligation reaction (Level 1)
![](https://static.igem.org/mediawiki/2014/0/01/DIGLIGLEVEL1.jpg)
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)
![](https://static.igem.org/mediawiki/2014/e/e2/Digliglevel2.jpg)
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
![](https://static.igem.org/mediawiki/2014/4/43/MAKINGLBAGAR.jpg)
Make up with distilled water, ensuring all solid is mixed thoroughly. Pour into appropriately sized Erlenmeyer flasks with 1.5% w/v agarose in each flask, place sponge stopper on top and autoclave.
Making LB broth
![](https://static.igem.org/mediawiki/2014/7/72/MAKINGLBBROTH.jpg)
Make up with distilled water, ensuring all solid is mixed thoroughly. Pour into appropriately sized Erlenmeyer flasks, place sponge stopper on top and autoclave.
E. coli Calcium Chloride Heat Shock Transformation
![](https://static.igem.org/mediawiki/2014/6/60/COLONYSTOCKS.jpg)
Aim: to get DNA expression in E. coli.
- Remove chemically competent E. coli (DH5-alpha) 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
Aim: to get DNA expression in E.coli.
Agrobacterium tumefaciens Electroporation Transformation
![](https://static.igem.org/mediawiki/2014/7/76/Agrocolonypcr.jpg)
Aim: to get DNA expression in Agrobacterium tumefaciens to allow infiltration into Nicotiana Benthamiana.
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
![](https://static.igem.org/mediawiki/2014/6/68/Colonypcragro.jpg)
Aim: to determine the size (bp) of the DNA plasmid expressed by the colony picked. This helps us to check that the construct is what we expected.
Component | Volume (µL) |
---|---|
dNTPs | 2.5 |
Primer 1 | 1.0 |
Primer 2 | 1.0 |
10x NH4 Reaction Buffer | 2.0 |
MgCl2 Solution (50mM) | 2.0 |
BIOTAQ DNA Polymerase | 0.5 |
Distilled water | 6.0 |
Step | Temperature (°C) | Time (minutes: seconds) |
---|---|---|
1 | 96 | 2:00 |
2* | 96 | 0.30 |
3* | 55 | 0.30 |
4* | 72 | 0.30 |
5 | 16 | ∞ |
DNA mini-prep
![](https://static.igem.org/mediawiki/2014/9/90/SEQUENCING.jpg)
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
![](https://static.igem.org/mediawiki/2014/4/4e/CARALAB.jpg)
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
![](https://static.igem.org/mediawiki/2014/2/2f/AGROINFILTRATION.jpg)
Aim: To get expression of our constructs in Nicotiana benthamiana leaves, allowing our system to be tested.
Infiltration Analysis
Aim: To analyse the expression of each of our constructs in Nicotiana benthamiana to allow us to see how well our system is working.
We used an epifluorescent microscope to view the results of our infiltrations into Nicotiana benthamiana.Making GoldenGate compatible PSB1C3 'Flipper'
![](https://static.igem.org/mediawiki/2014/4/4e/Labequipmentuea.jpg)
PROTOCOL HERE
*Antibiotic Selection
![](https://static.igem.org/mediawiki/2014/8/80/Antibiotic_selection.jpg)
The following antibiotic concentrations have been used for antibiotic selection throughout the project. Antibiotics have been added to agar plates and overnight cultures in order to select for bacteria carrying the desired plasmid.
Antibiotic | Use | Stock concentration | µL/ mL required |
---|---|---|---|
Chloramphenicol | PSB1C3 constructs | 33mg/ml | 1.5 |
Kanamycin | Level 0 constructs | 30mg/ml | 1 |
Carbenicillin | Level 1 constructs | 100mg/ml | 1 |
Ampicillin | Level 1 constructs | 100 mg/ml | 1 |
Spectinomycin | Level 2 constructs | ||
Rifampicin | Agrobacterium tumefaciens | 10mg/ml | 5 |
Gentamicin | Agrobacterium tumefaciens | 10mg/ml | 2 |