Biomedical Engineering Reference
In-Depth Information
2.3
PCR Reagents
1. 10× Pfu Ultra II PCR buffer (Stratagene).
2. 10 mM dNTP mixture.
3. Pfu Ultra II Fusion HS (Stratagene).
2.4 Gel
Electrophoresis
Components
1. UltraPure Agarose (Life technologies).
2. Agarose gel running chamber and power supply.
3. 25 mg/mL Ethidium Bromide stock solution.
4. 10× agarose gel loading buffer (2.5 % Ficoll 400, 11 mM
EDTA, 3.3 mM Tris-HCl, 0.017 % SDS, 0.015 % Bromophenol
Blue, pH 8.0 @ 25 °C).
5. GeneRuler DNA Ladder Mix (Fermentas).
6. UV illumination and camera system.
7. Agarose gel purifi cation kit (Qiagen).
2.5 Chew-Back
Assembly Reagents
1. 10× T4 DNA polymerase buffer: 100 mM Tris-HCl, 500 mM
NaCl, 100 mM MgCl
2
, 10 mM DTT, pH 7.9.
2. 1 mg/mL bovine serum albumin (NEB).
3. 20 mM dATP dissolved in H
2
O.
4. 20 mM dTTP dissolved in H
2
O.
5. 3 U/
μ
L T4 DNA polymerase (NEB).
1. Chemo-competent
E. coli
in aliquots of 50
μ
L, stored at
2.6 Bacterial
Components
g.
2. Lysogeny Broth (LB) medium (25 g powder dissolved in 1 L
water; autoclaved).
3. 100 mg/mL Ampicillin stock solution.
4. QIAprep Spin Miniprep Kit (Qiagen).
−80 °C, more than 10
7
cfu/
μ
1. 10× AarI buffer (Fermentas).
2. 2 U/
2.7 Enzymatic
Restriction Reagents
μ
L AarI (Fermentas).
3. 25
M AarI supportive oligonucleotide (Fermentas).
4. DpnI (Fermentas).
μ
1. pcDNA3.1 (Life technologies).
2.8
Plasmids
1. Ape Plasmid Editor (
http://biologylabs.utah.edu/jorgensen/
wayned/ape/
)
.
2.9
Software
2.10
Devices
1. PCR thermal cycler.
2. Spectrophotometer (NanoDrop 1000).
3. Thermoblock (Eppendorf Thermomixer comfort).
