Biomedical Engineering Reference
In-Depth Information
additional intermediate categories). Therefore we arbitrarily assigned
fl anking 4 nt to each of the three basic categories (
see
Fig.
1b
) that
have to be used in every GBpart to ensure compatibility between
different users.
Basic parts normally come in the form of a circular plasmid,
and the insert corresponds to the part itself. Upon BsaI digestion,
the part is released from the plasmid leaving 4 nt overhangs, ready
to be assembled together with other parts in a BsaI GB-reaction.
A large collection of ready-to-assemble standard GBparts has been
established and can be consulted at
http://www.gbcloning.org
.
1. Gene-specifi c Oligonucleotides with GB extensions (
see
Subheading
3.1
).
2. Phusion
®
High-Fidelity DNA Polymerase (ThermoScientifi c).
3. dNTP mixture (10 mM each dNTP).
4. Milli-Q sterilized water.
5. Biotools DNA Polymerase (Biotools).
6. Agarose electrophoresis gel: 1 % agarose TAE 1 × (40 mM
Tris-acetate and 1 mM EDTA).
7. QIAquick PCR Purifi cation Kit (Qiagen).
8. pGEM
®
-T Easy Vector System (Promega).
9. Thermocycler.
10. 50 % Glycerol for storing the correct assemblies.
2.3 Materials
for PCR Amplifi cation
and TA-Cloning of
DNA Pieces
1. House-made competent Cells, One Shot
®
TOP10 or One
Shot
®
Mach1™ T1R chemically competent
Escherichia coli
kit
(Invitrogen).
2. Electroporator and 1 mm electroporation cuvettes.
3. Sterile SOC medium: 2 % tryptone, 0.5 % yeast extract, 10 mM
sodium chloride, 2.5 mM potassium chloride, 10 mM magne-
sium chloride, 10 mM magnesium sulfate, 20 mM glucose.
4. Sterile Lysogeny Broth (LB) medium: 1 % tryptone, 0.5 % yeast
extract, 1 % NaCL.
2.4 Escherichia coli
Cell Transformation
and Culture
5. Lysogeny Agar (LA) plates: 1 % tryptone, 0.5 % yeast extract,
1 % NaCL, 1.5 % agar. Plates contain the appropriate antibiotics
(kanamycin at 50
μ
g mL
−1
, ampicillin and spectinomycin at
100
μ
g mL
−1
), IPTG (0.5 mM), and X-Gal (40
μ
g mL
−1
).
6. A shaker and growing chamber at 37 °C.
7. E.Z.N.A. Plasmid Mini Kit (Omega Bio-tek).
1. Miniprep-purifi ed GBparts.
2.
2.5 Materials
for Multipartite
Assembly Reactions
-level GB destination vectors.
3. T4 DNA ligase (Promega, Madison, USA), BsaI restriction
enzyme (New England Biolabs).
α
