Biomedical Engineering Reference
In-Depth Information
9. Pick one of the transformants from the plate and inoculate into
3-5 mL of the LB medium supplemented with appropriate
antibiotic and incubate at 37 °C for 10-12 h. Extract plasmid
by Plasmid Miniprep isolation kit.
10. Check the plasmid (Fig.
3a
, lane 5) and its digestion product
by restriction enzymes (Fig.
3a
, lane 6).
11. If necessary, the plasmid can be sequenced for further
validation.
3.3 DNA Assembly
This method can also be used to assemble three or more fragments
in one step (Fig.
4a
). For example, three pairs of primers (IF1/
IR1, IF2/IR2, and VF/VR) are used to amplify the two DNA
insert fragments and one vector backbone. Here fructose-1,6-
bisphosphatase (fbp, 0.80 kb) gene from
Thermotoga maritime
, a
dockerin module (docRF, 0.25 kb) from
Ruminococcus fl avefa-
ciens
, and pET20b backbone are assembled by POE-PCR.
1. The pET20b vector backbone is amplifi ed with a pair of prim-
ers of VF and VR through PCR by using the Phusion DNA
polymerase (Fig.
4b
, lane 1).
2. The
fbp
DNA fragment is amplifi ed with a pair of primers of
IF1 and IR1 by using the Phusion DNA polymerase (Fig.
4b
,
lane 2).
3. The
docRF
DNA fragment is amplifi ed with a pair of primers
of IF2 and IR2 by using the Phusion DNA polymerase (Fig.
4b
,
lane 3).
4. The three PCR products are cleaned with the DNA Clean &
Concentrator Kit.
5. Determine the DNA concentration of the three purifi ed DNA
fragments with the Nanodrop ND-1000 Spectrophotometer
or with agarose gel electrophoresis.
6. For POE-PCR, add the below reagents to the tube:
Component
Final concentration
5× Phusion HF or GC Buffer
10
μ
L
1×
dNTPs (10 mM each)
1
μ
L
200
μ
M each
Vector DNA fragment
Variable
4 ng/
μ
L
Insert DNA fragment 1
Variable
Equal molar with vector
Insert DNA fragment 2
Variable
Equal molar with vector
Water
To 49.5
μ
L
Phusion DNA polymerase
0.5
μ
L
1.0 unit per 50
μ
L
7. POE-PCR was conducted as described above for the generation
of DNA multimers.
