USER-Derived Cloning Methods and Their Primer Design - DNA Cloning and Assembly Methods - page 62

2 Materials

Unless stated otherwise use 18 m cm water in all buffers and

media and perform the procedures at room temperature.

Ω

2.1 PCRs

1. 10 mM dNTPs mixture (2.5 mM each of dATP, dCTP, dGTP,
and dTTP).
2. PfuTurbo ® Cx Hotstart DNA Polymerase (Agilent) or prefer-
ably the proof-reading X-7 polymerase [ 30 ].
3. 10× CxL polymerase buffer (200 mM Tris-HCl, 100 mM
KCl, 60 mM (NH 4 ) 2 SO 2 , 20 mM MgSO 4 , 1 mg/mL, 1 %
Triton X-100): Prepare 1 M stock concentrations of Tris-HCl
base, KCl, (NH 4 ) 2 SO 4 , and MgSO 4 in the following way.
Dissolve 15.756 g Tris-HCl base in 100 mL ddH 2 O and adjust
pH to 8.8 with NaOH ( see Note 1 ). Prepare three 100 mL
Blue cap bottles, one with 7.46 g of KCl, the second with
13.2 g of (NH 4 ) 2 SO 4 , and 22.8 g of MgSO 4 ·6H 2 O in the last
and fi ll to 100 mL with ddH 2 O. Mix 20 mL 1 M Tris-HCl
pH 8.8 with 10 mL of 1 M KCl, 6 mL of 1 M (NH 4 ) 2 SO 4 , and
2 mL of 1 M MgSO 4 . Add 10 mL of 100× BSA stock solution
(10 mg/mL, New England Biolabs) and 1 mL Triton X-100.
Fill to 100 mL with ddH 2 O, sterile fi lter, and store at −20 °C.
4. Template DNA.
5. 10 M primer solutions.

6. Enzyme (New England Biolabs): DpnI (20,000 units/mL).

μ

2.2 Preparation
of Plasmid with USER
Cassette

This step is not necessary when performing UCF-USER fusion.

1. Appropriate restriction- and nicking enzyme with buffers for
the digestion of the USER compatible vector ( see Note 2 ). For
example, the USER cassette in Fig. 1 should be digested with
PacI and Nt.BbvCI (New England Biolabs).

1. Enzyme (New England Biolabs): USER™ enzyme (1,000 units/mL).

2. Solid Lysogeny Broth (LB) media: Dissolve 5 g of yeast extract,
10 g of sodium chloride (NaCl), 10 g of tryptone, and 20 g of
agar in 1,000 mL ddH 2 O. Autoclave and add antibiotics for
selection.
3. Liquid LB media: Autoclave 1,000 mL ddH 2 O containing yeast
extract (5 g/L), sodium chloride (NaCl 10 g/L), and tryptone
(10 g/L). Add selective antibiotics after autoclavation.

2.3 USER Cloning

4. Chemically competent E. coli cells, e.g., TOP10 or DH10B.
5. Plasmid isolation: GenElute Plasmid Miniprep Kit (Sigma-Aldrich).