Biomedical Engineering Reference
In-Depth Information
3. Microcentrifuge capable of 14,000 ×
g.
4. Sterile 1.5 mL microcentrifuge tubes.
5. Centrifuge capable of 10,000 ×
g.
2.4 Restriction
Digestion of BioBricks
1. Bucket with ice.
2. Microcentrifuge tubes, 1.5 mL.
3. Distilled H
2
O.
4. NEB Buffer 1, 2, 3, and 4.
5. Bovine serum albumin (BSA).
6. Restriction enzymes: EcoRI, SpeI, XbaI, PstI (for BBF RFC
10, if another assembly standard is used, other enzymes might
be required).
7. Water bath or thermal cycler, 37 and 80 °C.
8. Spectrophotometer (e.g., NanoDrop).
2.5 Gel
Electrophoresis
1. Agarose gel (low EEO, for less smeared bands) with Gel Green™.
2. TBE buffer (Tris/Borate/EDTA).
• Preparation of stock solution: 54 g tris base, 27.5 g borate,
20 mL 0.5 M EDTA, diluted up to 1 L with dH
2
O.
• For use: dilute 100 mL with 900 mL distilled H
2
O.
3. Loading dye (0.2 volume of loading dye, e.g., 4-20 μL).
• Example recipe: 25 mg bromophenol blue, 4 g sucrose,
diluted with dH
2
O to 10 mL. If using bromophenol blue,
be aware that it migrates equally quickly as 200-400 bp
sized DNA pieces, and will therefore hide those fragments.
In cases where this applies, use another dye.
4. 1 kb DNA ladder.
5. Gel documentation unit.
1. QIAquick Gel Extraction Kit (Qiagen).
2. Pieces of gel with DNA.
3. Ethanol (96-100 %).
4. Isopropanol (100 %).
5. Heating block or water bath, 50 °C.
6. Microcentrifuge capable of 14,000 ×
g.
2.6
Gel Purification
1. Digested BioBricks to be ligated (the amount of digested DNA
is determined by expression 1).
2. T4 DNA Ligase Reaction Buffer.
3. T4 DNA Ligase.
4. Heating block, 16 and 80 °C.
2.7
Ligation
