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CH4.3 Stage 3 – Make your BioBrick

Here are the descriptions of one of the traditional way to make a BioBrick. General materials are listed to be prepared before the lab starts, and some points should be noted in the process.

This session can be used as your check list before you are familiar with the actual protocol from different kits, but please take note that variation always exists and understanding the protocol before start is helpful as well as necessary for the desired result.

Do not be upset with negative result. Protocols vary among the different designs of the BioBrick, and asides from the one described above, there are other ways to make a BioBrick.

Part 1: BioBrick Construction

Step 1: Polymerase Chain Reaction (PCR)

A DNA amplification process to make the target sequence into linear DNA copies

Material: dNTPs, water, DNA templates, forward and reverse primers, heat stable DNA polymerase and its corresponding buffer

Note:
 DNA templates should be added at last, and thermocycle should start immediately after adding all the materials

Step 2: DAN Digestion by Restriction Enzyme (Restriction Cut)

Restriction cut can be used and repeated for different purposes: create a sticky end of the DNA, make blunt end of DNA, or make linear plasmid into circular form.

Material: water , DNA template, restriction enzyme, 100X BSA buffer, 10X NEWB buffer, and 37。C incubator or dry bath

Note:
 Water is added first
 Buffer is added before enzyme
 Ensure the pipette tip touches the surface of solution
 Glycerol can be added to prevent further undesirable DNA digestion

Step 3: DNA Purification

Removing excess nucleotides, enzymes and ions in the solution with desired 100 bp - 10 kb DNA

Material: use PCR Purification Kit

Note:
 Elution efficiency is pH dependent, and the optimal pH for maximum efficiency is in between pH 7 and 8.5
 DNA should be stored at 20 ° C to prevent degradation
 EDTA buffer may inhibit subsequent enzyme reactions
 Recovery can be maximized by warm elution buffer

Source: Loo J. 2012. iGEM Hong_Kong-CUHK 2012 Wet-lab Training Manual.
The Chinese University of Hong Kong School of Life Science.Version 2.2.

Step 7: Biobrick Amplification in Plasmid Form

Amplifying the desire DNA plasmid by growing the colonies from the spread plates with antibiotic to select the expect bacteria which contains the backbone making the bacteria to be resistant to a particular antibiotic.

Material: autoclaved tooth pick, LB media, antibiotic, snap cap, and 37。C shaker

Notes:
 Require aseptic technique for picking clone
 Mark the clone you picked
 Storage of the bacteria cell with desired plasmid: 800 μl of cell + 200 μl of 80% sterile glycerol, and they should be stored in -80。C freezer.

Step 8: Plasmid DNA Extraction

Material: miniprep plasmid DNA extraction kit

Notes:
 Be careful when transferring supernatants from centrifuge tubes into the spin column to avoid contamination of genomic DNA
 To increase the concentration of plasmid, increase the incubation time while the Elution buffer stains in a 60 。C dry bath

">Source: Loo J. 2012. iGEM Hong_Kong-CUHK 2012 Wet-lab Training Manual.
The Chinese University of Hong Kong School of Life Science.Version 2.2.

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