Biomedical Engineering Reference
In-Depth Information
6. Transfer a colony to liquid medium (Subheading
3.2
).
7. Miniprep the transformants (Subheading
3.3
), to obtain
plasmid DNA.
3.9 Amplified Insert
Assembly Protocol
The protocol given here is taken from the OpenWetWare website [
19
].
1. Miniprep both insert and plasmid from their respective cultures
(
see
Subheading
3.3
).
2. Amplify the insert using PCR.
●
Use a high-fidelity polymerase
●
Primers:
- The primers should flank the restriction sites by
100-150 bp.
- The primers should have a
T
m
of 55-60 °C.
- For most Biobrick applications, the primers VF2 and
VR can be used.
●
Run 25-30 cycles, as this will help ensure high fidelity.
3. Start the vector digestion while the PCR is still running. Digest
the vector with the appropriate restriction endonucleases for
2 h (
see
Subheading
3.4
).
4. Purify the PCR product (
see
Subheading
3.6
).
5. Digest the purified insert for 1 h with enzymes complementary
to the vector digest. Include DpnI in the reaction mixture
(
see
Subheading
3.4
).
6. Add 6 μL Antarctic Phosphatase Buffer and 1 μL Antarctic
Phosphatase to the vector digest, and incubate until the insert
digest is done.
7. Heat inactivate all enzymes by incubating the reaction mixtures
for 20 min at 80 °C.
8. Ligate at a molar ratio of 4:1 (insert:vector) (
see
Subheading
3.7
).
9. Transform the ligation mixture to competent cells (
see
Subheading
3.1
).
10. Plate out the transformed cells on agar plates containing a
suitable antibiotic.
11. Celebrate!
4
Software Tools
4.1 Sources of
Software Developed
Within the iGEM
Competition
Starting in 2008, awards for software tools were introduced in the
iGEM competition [
20
]. The efforts of iGEM teams have since
then produced a number of software tools performing a wide vari-
ety of tasks that are relevant when working with BioBrick parts
from the Registry of Standard Biological Parts. The latest tools that
have been created can be located through the iGEM repository at
