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Revision as of 16:59, 18 September 2015

Protocol

(1) Bacterial DNA extraction protocol for Azotobacter vinelandii or E.coli

- We are using the TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit of Takara according to the manufacturer directions.

(2) Miniprep

- We are using the DNA-spin™ Plasmid DNA Purification Kit of Intron Technology according to the manufacturer directions.

(3) Preparation of chemically competent BL21 E.coli cells

Day1

  • Streak Bl21 on a LB agar plate without antibiotic, grow overnight in 37℃ incubator

Day2

  • Pick a single colony and inoculate into 3ml LB broth, grow o/n in 37℃ shaker. Prepare & autoclave 500ml LB broth. Check if there is enough liquid nitrogen.

Day 3 (morning)

- Pour the 3ml dense pre-culture into 500ml LB broth. Shake in 37℃ until OD 600nm reach 0.8 [it takes 4-5 hours by experience]

- Solution needed:

-Wash Buffer I (800mM MgCl2 + 20mM CaCl2)

-Wash Buffer II (125mM CaCl2)

-Re-suspension Buffer (85mM CaCl2 + 15% glycerol [filtered])

1. pre-cool Wash Buffer I & Wash Buffer II in ice

2. Pre-cool the centrifuge to 4C (with fixed angle rotor)

3. Check the OD600nm of the 500ml culture

4. Centrifuge the cells at 4000g for 5 mins, 4℃

5. Discard the supernatant

6. Gently resuspend the pellet in 20ml ice cold Wash Buffer I

7. Put the samples on ice for 10 mins

8. Centrifuge the cells at 4000g for 5 mins, 4℃

9. Discard the supernatant

10. Gently resuspend the pellet in 10ml ice cold Wash Buffer II

11. Put the samples on ice for 10 mins

12. Centrifuge the cells at 4000g for 5 mins, 4℃

13. Discard the supernatant

14. Resuspend cells in 20ml ice cold Resuspend Buffer

15. Aliquot 200ul using sterile pre-chilled eppendorf tubes

========================================================================

(4) Primer Design

Primers were designed manually using Snapgene

========================================================================

(5) PCR - Phusion DNA polymerase NEB

For a 50ul reaction

Reactives Volume(μL)
DNA template 1
5x Phusion HF Buffer 10
10mM dNTPs 1.5
10uM Primer Fw 0.5
10uM Primer Rv 0.5
Phusion DNA polymerase 0.25-0.5
100% DMSO 1.5
dH20 Up to 50
Total: 50

Cycling condition:

Initial Denaturation (1 cycle) 98°C -- 30 sec
Amplification (35 cycles)

98°C -- 10 sec

55-72°C -- 30 sec

72°C -- (0.25-0.5 min/kb)

Final elongation (1 cycle) 72°C -- 3 min.
========================================================================

(6) PCR - LA Taq DNA polymerase Takara

For a 50ul reaction

Reactives Volume(μL)
DNA template 1
10x LA taq Buffer 5
10mM dNTPs 1.5
10uM Primer Fw 0.5
10uM Primer Rv 0.5
LA taq polymerase 0.25-0.5
100% DMSO 1.5
dH2O Up to 50
Total: 50

Cycling condition:

Initial Denaturation (1 cycle) 94°C -- 30 sec
Amplification (30 cycles)

98°C -- 10 sec<\p>

55-72°C -- 30 sec <\p>

68°C -- (0.5-1 min/kb) <\p>
Final elongation (1 cycle) 72°C -- 5 min.
========================================================================

(7) Double digestion of DNA with 2 different restriction enzymes NEB

30μl reaction

Reactives Volume(μL)
DNA Up to 1 μg
NEB Buffer 2 3
EcoRI/Xbal/SpeI/PstI 1
EcoRI/Xbal/SpeI/PstI 1
dH20 Up to 30 μL
Total: 30

Incubate at 37 °C incubator or heat bath for 0.25 to 2 hours.

*For combinations of restriction enzymes other than the above, please kindly refer to Double Digest Finder from NEB Inc. for suitable buffer condition.

========================================================================

(8) DNA ligation with T4 DNA ligase NEB

Reactives Volume(μL)
T4 DNA ligase 1
Buffer 10X 2
Vector DNA n pmol
Inesrt DNA 3n pmol
dH20 Up to 20 μL
Total: 20

Incubate at room temperature for 0.25 -1 hours.