Biomedical Engineering Reference
In-Depth Information
of 2
L of
10× buffer, and ultrapure H
2
O to a fi nal volume of
100
ʼ
g of DNA sample, 1
ʼ
L of restriction enzyme, 10
ʼ
L. Incubate the mixture at 37 °C for 3 h.
5. Add 10
ʼ
L of 3 M sodium acetate (pH 5.2) to a fi nal concen-
tration of 0.3 M and mix thoroughly.
6. Add 275
ʼ
L (2.5 volumes) of 99.5 % ice-cold ethanol and mix
thoroughly.
7. Store the solution at −80 °C for 10 min and centrifuge at
15,000 ×
g
for 10 min at 4 °C.
8. Discard the supernatant and add 500
ʼ
ʼ
L of 70 % ice-cold
ethanol.
9. Centrifuge at 15,000 ×
g
for 5 min at 4 °C and discard the
supernatant.
10. Dry the DNA pellet in a lyophilizer.
11. Dissolve the DNA pellet in 10
L of autoclaved Milli-Q water
and subject it to 1 % agarose gel electrophoresis. Cut out the
area of the gel containing the DNA fragments and extract
the DNA using QIAEX II Gel Extraction Kit, according to the
manufacturer's instructions.
12. DNA ligation using Ligation High or analogous DNA ligases:
Combine 20-400 fmol of DNA fragment, 50-100 fmol of
plasmid DNA, an appropriate amount of DNA ligase, and
ligase buffer. Incubate the mixture at 16 °C for 1 h and follow
the manufacturer's protocols.
13. Transform 5
ʼ
L
of ligation reaction and plate onto an LBA plate using a fl ame-
sterilized spreader. Incubate the plates at 37 °C overnight.
14. Inoculate 5 mL of the LBA media with
E. coli
transformants.
Incubate at 37 °C with overnight shaking.
15. Extract and purify the plasmid DNA using a miniprep kit (e.g.,
Quantum Prep Plasmid Miniprep Kit), according to the manu-
facturer's instructions. Determine the concentration and purity
of the extracted plasmid DNA from the absorbance measured
at 260 and 280 nm, using a spectrophotometer.
16. Verify the identity of the plasmid DNA constructs by restric-
tion digestion using appropriate enzymes. Digest 2
ʼ
L of
E. coli
DH5
ʱ
competent cells with 1.5
ʼ
g of
plasmid DNA as described in
step 4
and perform electropho-
resis on a 1 % agarose gel.
17. Perform sequencing on the ligated DNA fragment by using a
forward primer that anneals to the upstream region (GAPDH
promoter) of the multiple cloning sites (pGAP_F; 5
ʼ
′
-AGA
CGGTAGGTATTGATTGTAATTCTG-3
) and appropriate
primers that can anneal to the internal sequences of the ligated
DNA fragment.
′
