Biomedical Engineering Reference
In-Depth Information
Table 2
T4 DNA polymerase digestion and ligation
T4 DNA polymerase digest
Fragment
(28 μL)
Conc.
(ng/μL)
Total DNA
(ng)
T4 Pol
(0.5 U/μL) (μL)
Buffer
(μL)
dCTP
(10 mM) (μL)
Final vol.
(μL)
Final conc.
(ng/μL)
1
54
1,512
1.5
3.3
3.6
36
42
2
45
1,260
1.3
3.3
3.6
36
35
3
83
2,324
2.3
3.4
3.7
37
62
4 (plasmid)
55
1,540
1.5
3.3
3.6
36
42
Ligation
Fragment
Conc. (ng/
μ
L)
Size (kb)
Amount (ng)
Volume (
μ
L)
1
42
3.80
108
2.6
2
35
1.96
56
1.6
3
62
0.89
25
0.4
4 (plasmid)
42
5.27
150
3.5
Lig. Buffer
0.9
RecA (optional)
0.1
(200 ng/
μ
L)
Total
9.1
(kb). This is then multiplied by 150 ng to give the required
amount (ng) of insert (
see
Table
2
for an example). Finally add
one-tenth volume 10× ligation buffer. Incubate at 37 °C for
30 min (
see
Note 15
).
5. Place the “ligation” on ice for 5 min and then transform the
entire reaction into competent
E. coli
cells, which have a trans-
formation effi ciency of >10
6
colonies per
g of DNA. Follow
the transformation procedure provided with the competent
cells (be it heat-shock, electroporation, etc.). Plate 150
μ
L of
cells onto one agar selection plate, concentrate the remaining
cells to about 150
μ
μ
L, and plate onto a second selection plate
(
see
Note 16
).
3.4 Screening for
Correct Assembly
1. Assuming transformation was successful, screen 5-15 colonies
via colony PCR utilizing primers used in construction of the
plasmid. Use a forward primer from one fragment and a reverse
primer from a 3
adjacent fragment. Colony PCR uses the
same reaction mix as described in Subheading
3.2
; however,
′
