Biomedical Engineering Reference
In-Depth Information
2. Restriction endonuclease BsaI (10 U/
μ
L) (NEB), supplied with
3. Restriction endonuclease BpiI (10 U/
10× NEBuffer 4.
L) (Fermentas GmbH,
St. Leon-Rot), supplied with 10× Buffer G (100 mM Tris-HCl
pH 7.5, 100 mM MgCl
2
, 500 mM NaCl, 1 mg/mL BSA).
μ
4. T4 DNA Ligase 3 U/
L
(Promega, Mannheim), both supplied with 10× ligation buffer
(300 mM Tris-HCl pH 7.8, 100 mM MgCl
2
, 100 mM DTT,
10 mM ATP).
5. For measuring of DNA concentration, we use the NanoDrop
ND2000 (Peqlab, Erlangen).
6. Lysogeny Broth (LB) Medium: 1 % bacto-tryptone, 0.5 % yeast
extract, 1 % NaCl in deionized water, adjusted to pH 7.0 with
5 N NaOH. For plates, 1.5 % agar is added.
μ
L or T4 DNA Ligase (HC) 20 U/
μ
7. Antibiotics carbenicillin (used instead of ampicillin) and kana-
mycin: fi lter-sterilized stocks of 50 mg/mL in H
2
O (stored in
aliquots at −20 °C) are diluted 1:1,000 (fi nal concentration:
50
g/mL) in an appropriate amount of medium after the
medium has been autoclaved and cooled down. For spectino-
mycin, a stock of 40 mg/mL is made and is used at a fi nal
concentration of 100
μ
g/mL (dilution 1:400).
8. 5-Bromo-4-chloro-3-indolyl-
μ
-
D
-galactopyranoside (X-gal):
stock solution of 20 mg/mL in dimethylformamide (DMF).
For preparation of plates, the stock is diluted 1:500 (fi nal
concentration: 40
β
g/mL) in an appropriate amount of LB
agar after autoclaving/melting and cooling down.
μ
1. NucleoSpin
®
Plasmid Quick Pure (Macherey Nagel, Düren)
for preparation of miniprep DNA.
2. Restriction endonucleases (NEB or Fermentas), all supplied
with 10× buffer and if necessary also with 100× BSA (dilute
1:10 and store in aliquots at −20 °C).
3. DNA ladder: GeneRuler™ 1 kb DNA Ladder Plus (Fermentas)
is used as marker for gel electrophoresis.
4. 50× TAE buffer: 242.0 g Tris, 57.1 mL acetic acid, and
100 mL 0.5 M EDTA, pH 8.0, in 1 L of deionized water.
5. For preparation of gels for electrophoresis, agarose (0.7-1.5 %)
in 1× TAE is melted in a microwave oven and one drop of a
0.025 % ethidium bromide solution (Carl Roth GmbH,
Karlsruhe) is added per 100 mL of melted agarose.
6. Running buffer for agarose gels is 1× TAE.
7. Gels are checked visually using a Syngene GelVue transillumi-
nator (VWR, Darmstadt), and pictures are taken by using a
Quantity One
®
gel analysis software (Biorad).
8. DNA maps of plasmids are made by using the Vector NTI
software (Invitrogen).
2.3 Screening
of Colonies
