Seamless Ligation Cloning Extract (SLiCE) Cloning Method - DNA Cloning and Assembly Methods - page 233

11. 37 °C shaker.
12. 37 °C incubator.
13. Centrifuge.
14. Spectrophotometer.
15. −20 and −80 °C freezer.

2.2 Maintenance
of PPY strain

1. PPY strain.
2. Lysogeny Broth (LB) medium: Dissolve 10 g Bacto tryptone,
5 g Bacto-yeast extract, and 10 g NaCl in 900 mL ddH 2 O.
Adjust pH to 7.2 with NaOH. Adjust to 1 L with ddH 2 O.
Autoclave to sterilize and store at room temperature.
3. Antibiotics: Streptomycin and chloramphenicol.
4. 20 % Glycerol.
5. 37 °C shaker.
6. −80 °C freezer.

1. PPY SLiCE extract (prepared as described in Subheading 3.1 ).
2. 10× SLiCE buffer: 500 mM Tris-Cl (pH 7.5 at 25 °C),
100 mM MgCl 2 , 10 mM ATP, 10 mM DTT, store at −20 °C.
3. Materials and equipment for restriction cutting, PCR, DNA
purifi cation and transformation.

2.3 SLiCE Cloning

3 Methods

3.1 Preparation of
PPY SLiCE Extract

1. Streak PPY glycerol stock or fresh culture on an LB agar plate
(10 μ g/mL streptomycin and 12.5 μ g/mL chloramphenicol)

and incubate at 37 °C overnight.
2. Inoculate one single colony into a 50-mL centrifuge tube con-
taining 25 mL 2XYT (10 g/mL streptomycin) and shake at

37 °C and 330 rpm overnight.
3. The next day, measure the OD 600 ( see Note 2 ).
4. Dilute the o/n culture to 0.03 OD 600 , i.e., inoculate appropri-
ate volume of the o/n culture into a 250-mL Nalgene Lab
Quality Wide-Mouth Bottle containing 50 mL 2XYT medium
(10

μ

g/mL streptomycin) ( see Note 3 ).
5. Shake at 37 °C and 330 rpm until the culture reaches an OD 600
of 5.0-5.5 ( see Note 4 ).
6. Add 0.2 % L -(+)-arabinose into the culture and continue
shaking at 330 rpm for 2 h at 37 °C, to induce expression of

μ

λ

prophage protein Red. Remove 500 μ L from the culture to

measure the actual OD 600 .
7. Transfer 48 mL of the bacterial culture into two 50-mL
centrifuge tubes (24 mL each).