LB Basic Recipe: 10 g tryptone, 5 g yeast extract and 5 g NaCl per litre reverse osmosis water
- Make 2 litres by doubling above recipe. Divide into 4 x 400 ml, 1 x 200 ml in 500 ml bottles.
To make LB broths:
- Divide up one 400 ml broth further into 8 x 50 ml in glass 250 ml flasks, cover with foil. Sterilise by autoclaving alongside the 200 ml LB aliquot (still in its bottle).
- Allow to cool. Working aseptically, divide up the 200 ml broth into 40 x 5 ml aliquots in 50 ml Falcon tubes, organise in racks, label as ‘LB’ and the date. Ensure labels are visible also on the 50 ml broth flasks. These can all be stored on bench at room temperature.
To make LB agar:
- Take the other remaining 3 x 400 ml of LB broth from above, and add agar to each bottle to the recommended amount (15 g / L = 6 g / 400 ml). Autoclave. Transfer from autoclave to hot oven (60°C) while still hot. Allow to cool to 60°C.
- To two of the bottles, add 400 µl of kanamycin stock solution (50 mg/ml) to give a final conc. of 50 µg/ml Km in the agar. This will make two sleeves of LB-Km plates (~2 x 20 plates). Pour the plates in the biosafety cabinet. Label the side of each stack of plates at the end with a single green coloured stripe – this indicates LB-Km. Put back in sleeve, store at 4°C.
- To another bottle, add 400 µl of kanamycin stock solution (50 mg/ml) and 400 µL of IPTG stock solution (1 M) to give final concs. of 50 µg/ml Km and 1 mM IPTG in the agar. This will make one sleeve of LB-Km-IPTG plates (~20 plates). Pour the plates in the biosafety cabinet. Label the side of the stack of plates at the end with one green and one orange stripe – this indicates LB-Km-IPTG. Put back in sleeve, store 4°C.
TE: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. Make in reverse osmosis water. Sterilise by autoclaving.
PBS: 8 g NaCl, 1.44 g Na2HPO4, 0.245 g KH2PO4 and 0.2 g KCl per litre of reverse osmosis water, pH 7.4. Sterilise by autoclaving.
CCMB solution for competent RARE E.coli cells: 102 g CaCl2.H20, 0.4 g MnCl2.4H20, 0.2 g MgCl2.6H20, 0.5 g KAC, 10mL glycerol and 90 mL reverse osmosis water. Add salts to water, then add glycerol and filter sterile into falcon tubes.
PCR of pUS250 DNA Plasmid
1. Retrieve the following PCR reagents from the freezers. Thaw all reagents briefly, mix by flicking or vortexing and then put on ice.
- Phusion HF 2x Master Mix
- Primer NVC375 (50 µM)
- Primer NVC376 (50 µM)
- Template DNA
- Sterile MilliQ water
2. Make a 1/100 dilution of the plasmid template (1 µl DNA in 100 µl MQ) for use as the PCR template; keep this on ice. Return the concentrated stock of plasmid to the freezer.
3. Obtain four sterile PCR tubes. Set up the master mix in one of these tubes, as below.
- 100 µl of 2x Master Mix
- 2 µl of NVC375
- 2 µl of NVC376
- 2 µl of dilute template DNA
- 94 µl of MilliQ water
4. Return all source reagents to freezer. Split the 200 µl master mix into 4 x 50 µl between the PCR tubes, ensuring each tube contains 50 µl. Thermocycle according to the below scheme:
- Initial Denaturation: 98°C for 30 sec
- Then 30 cycles of: 98°C for 10 sec, 65°C for 10 sec, 72°C for 30 sec
- Final extension: 72°C for 5 min
- Then hold at: 15°C
5. Run out 3 µL of the PCR product from one of the tubes on an agarose gel to check for a product. Compare any bands to marker ladder (use 5 µL of pre-made DNA marker). There should be a single strong band at approximatelt 5 kb.
6. If the PCR is successful, pool the products together into one tube and add DpnI enzyme (1 µl). Incubate at 37°C for 15 min. Column-purify the mixture using the NEB kit.
7. Quantify the purified DNA using Nanodrop.
Golden Gate Reactions
1. Add the reagents (listed in the table below) and mix by gentle flicking.
| Reagents (PMQ3A) |
Amount (μl) |
|
Reagents (PMQ3B) |
Amount (μl) |
| ECH Ppu |
1.8 |
ECH Ppu |
1.8 |
| FCS Ppu |
3.6 |
FCS Ppu |
3.6 |
| ComT aru |
2.3 |
ComT 6714 |
2.3 |
| SAM5 |
3.4 |
SAM5 |
3.4 |
| SAM8 |
3.4 |
SAM8 |
3.4 |
| PSU250 |
1.1 |
PSU250 |
1.1 |
| 10x Ligase Buffer |
2 |
10x Ligase buffer |
2 |
| MilliQ Water |
1.4 |
MQW |
1.4 |
| GRG Enzyme |
1 |
GRG enzyme |
1 |
| Total |
20 |
Total |
20 |
| Reagents (PMQ3C) |
Amount (μl) |
|
Reagents (PMQ3D) |
Amount (μl) |
| ECH Ppu |
1.8 |
ECH str |
1.8 |
| FCS Ppu |
3.6 |
FCS str |
3.6 |
| ComT aru |
2.3 |
ComT ara |
2.3 |
| hpaCB |
4.4 |
SAM5 |
3.4 |
| SAM8 |
3.4 |
SAM8 |
3.4 |
| PSU250 |
1.1 |
PSU250 |
1.1 |
| 10x Ligase buffer |
2 |
10x Ligase buffer |
2 |
| MQW |
0.4 |
MQW |
1.4 |
| GRG enzyme |
1 |
GRG enzyme |
1 |
| Total |
20 |
Total |
20 |
2. Incubate in a thermocycler according to the following conditions: 37°C for 5 mins, 16°C for 5 mins and 60°C for 5 mins, for x30 cycles. If planning to leave overnight, add a 4°C hold step after 30 cycles. Before using the DNA for transformation, incubate at 60°C for 5 mins.*
* An alternative shortcut method involves incubating the mixture at 37°C for 40 mins.
Transformation
1. Add 5 µl of ligation mixture to 50 µl of competent DH10B cells. Incubate on ice for 10 min, heat shock at 42°C for 45 sec, place back on ice 2 min, add 1 ml of LB broth and put at 37°C for 1 hr.
2. Mix by vortexing, then pipette 100 µl onto LB-Km-cumate agar, spread plate with glass beads. Centrifuge the remaining culture (2 min at 16,000 g) then pour off most of the supernatant. Resuspend the cells by vortexing in the last drop of liquid remaining, then plate that culture onto a second LB-Km plate.
3. Incubate plates at 37°C for 16-24hr. Recombinants should form white colonies.
Digestion and Ligation
1. Make a digestion mixture.
2. Mix by gentle flicking and incubate the digest at 37°C for 30 min. Stop with heat (80°C for 20 min).
3. Add ligase buffer and Hi-T4 ligase enzyme to the above digest, mix by gentle flicking and incubate at 37°C for 60 min.
