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Revision as of 23:51, 1 November 2017
protocols
Transformation with Digest
- DNA miniprrep
- LB agar plates, Cat No. Teknova, appropriate antibiotic
- DH5a competent cells, Invitrogen, Cat 18265-017
- SOC (Recovery Medium), Lucigen, Cat No. F98226
- 1.5 mL tube
- Vortex
- Pipet and tips
- Ice bucket and ice
- Water bath (42°C)
- Incubator (37°C) and shaker
- Transform 1 µL of DNA mini-preparation into E coli BL21 DE3 chemically competent cells
- For double transformation, transform with of 1 µL of each DNA mini-preparation into E coli BL21 DE3 chemically competent cells
- Turn on incubator-shaker at 37°C.
- Turn on incubator for plates at 37°C.
- Set up water bath at 42°C.
- Bring to room temperature S.O.C medium.
- Bring LB plates supplemented with appropriate antibiotic at room temperature.
- Thaw competent cells on ice.
- Aliquots competent cells in as many tubes as needed.
- Add 1.0 µl DNA preparation to 40 µl competent cells to DNA and swirl gently to mix
- Incubate on ice for 20 minutes
- Heat shock at 42°C in heat block for 30 seconds. Quickly return to ice and let it sit for 2 min.
- Add 200 µl of 18-25°C SOC medium and transfer mixture to 15mL Falcon tube
- Incubate in shaker at 37°C, 225 rpm for 30 min
- Plate 1 volumes (~100µL) of the mixture onto one LB agar plates supplemented with appropriate antibiotic: (for us Chloramphenicol 35 µg/mL or Tetracycline 12.5 µg/mL or both Chloramphenicol 35 µg/mL and Tetracycline 12.5 µg/mL depending on the clone)
- Incubate plates at 37°C overnight
- Initiate cultures in LB with the appropriate antibiotic from individual clones to analyze clones by digestion
Digest Ligations
- T4 DNA Ligation Buffer. Invitrogen, Cat No. 46-0114
- T4 DNA Ligase, Thermo Scientific, Cat No. K1231
- Reagent grade water, NERL, Cat No. 98555
- 1.5 mL tube
- Vortex
- Pipet and tips
- Ice bucket and ice
- Set ligation as shown below in 1.5 mL tube.
- The tubes were incubated at room temperature for 1 hour.
- Turn on incubator-shaker at 37°C.
- The tubes were then transferred to ice.
- Ligation Condition (with insert)
Component Volume (μl)
Reagent grade water 4.5 μl
10X T4 ligation buffer 1.0 μl
EcoR1-Pst1
Linearized - pSB1C3 0.5 μl
EcoR1/SpeI - Linearized –promoter
(For us: BBa_J23100, BBa_J23101, BBa_J23110) 2.0 μl
XbaI/Pst1 - Linearized -Insert (Catabolic pathway)
(For us: Fluorene 1, or Fluorene 2, or Phenanthrene 1, or Phenanthrene 2)
1.5 μl
T4 DNA Ligase (5Weiss/μl) 1.5 μl
- Control Ligation Condition (no insert)
Component Volume (μl)
Reagent grade water 8.0 μl
10X T4 ligation buffer 1.0 μl
EcoR1-Pst1 pSB1C3 0.5 μl
T4 DNA Ligase (5Weiss/μl) 0.5 μl
Component | Volume (μl) |
---|---|
Reagent grade water | 4.5 μl |
10X T4 ligation buffer | 1.0 μl |
EcoR1-Pst1 Linearized - pSB1C3 | 0.5 μl |
EcoR1/SpeI - Linearized –promoter (For us: BBa_J23100, BBa_J23101, BBa_J23110) | 2.0 μl |
XbaI/Pst1 - Linearized -Insert (Catabolic pathway) (For us: Fluorene 1, or Fluorene 2, or Phenanthrene 1, or Phenanthrene 2) | 1.5 μl |
T4 DNA Ligase (5Weiss/μl) | 1.5 μl |
Component | Volume (μl) |
---|---|
Reagent grade water | 8.0 μl |
10X T4 ligation buffer | 1.0 μl |
EcoR1-Pst1 pSB1C3 | 0.5 μl |
T4 DNA Ligase (5Weiss/μl) | 0.5 μl |