Biomedical Engineering Reference
In-Depth Information
4. SB medium: Superior Broth (AthenaES, Baltimore, MD),
40 g/L, autoclave for 20 min.
5. LB agar plates: Lysogeny Broth medium (10 g/L tryptone,
5 g/L yeast extract, 5 g/L NaCl) with 0.7 % agar, autoclave
for 20 min, cool to 55 °C, and add antibiotics as needed.
6. Kanamycin (Sigma, St. Louis, MO), 50
g/mL fi nal concen-
tration; stock solution 50 mg/mL in water, sterile fi lter, store
at 4 °C.
7. Spectinomycin (Sigma, St. Louis, MO), 100
μ
g/mL fi nal con-
centration; stock solution 100 mg/mL in water, sterile fi lter,
store at 4 °C.
8. Falcon 2059 culture tubes (Fisher Scientifi c, Pittsburgh, PA).
9. Supercoiled DNA Ladder (Life Technologies Corporation,
Carlsbad, CA). Store at 4 °C.
10. Temperature-controlled water bath.
11. Tabletop centrifuge.
μ
2.4 LR Multisite
Recombination
1. LR Clonase II enzyme mix (Life Technologies, Carlsbad, CA;
comes with LR Clonase
®
II enzyme mix, 2
μ
g/mL Proteinase
K solution).
2. Ampicillin (Sigma, St. Louis, MO), 100
g/mL fi nal concen-
tration; stock solution 100 mg/mL in water, sterile fi lter, store
at 4 °C.
3. QIAprep
®
Spin Miniprep Kit (Qiagen Inc., Valencia, CA).
4. BsrGI restriction enzyme (New England Biolabs,
Beverley, MA).
μ
1.
E. coli ccdB
Survival competent cells (Life Technologies,
Carlsbad, CA). Store at −80 °C; do not reuse open vials.
2. EcoRV restriction enzyme (New England Biolabs,
Beverley, MA).
3. DNA Polymerase I, Klenow fragment (New England Biolabs,
Beverley, MA).
4. T4 DNA Polymerase/Quick Blunting Kit (New England
Biolabs, Beverley, MA).
5. T4 QuickLigase (New England Biolabs, Beverley, MA).
6. QIAquick PCR Purifi cation Kit (Qiagen, Valencia, CA).
7. ReadyLoad 1 kb DNA ladder (Life Technologies,
Carlsbad, CA).
8. Temperature-controlled water bath.
9. Chloramphenicol (15 mg/mL stock in 100 % ethanol).
2.5 Destination
Vector Construction
