Biology Reference
In-Depth Information
4.
Mix 50
m
L of competent
E
.
coli
DH5
a
cells (prepared by the method of
Hanahan,
L ligation mixture and incubate on ice for 1 h. Pulse heat at 42
C
for 90 s before adding 150
1983
) with 5
m
L of LB. Incubate at 37
C for 1 h and then plate onto
nutrient agar containing the appropriate antibiotic.
5.
Screen colonies using a forward primer that anneals 50 nt upstream and a reverse
primer that anneals 50 nt downstream from the MCS or ligation-independent
cloning (LIC) site. This will generate a band of 100 bp if cloning is unsuccessful
and a band of the gene fragment size plus 100 bp if cloning is successful. The
positions of these primers also make them ideal for sequencing the inserted DNA
once positive clones have been identified.
2.1.2
Cloning into pYG1
1.
Cloning into pYG1 using LIC requires generation of long single-stranded
overhangs, and consequently, PCR primers containing 5
0
-
GGGTTCCTGGCGCGAGC-3
0
at the 5
0
-end of the forward primer and 5
0
-
TTGGGCTGGCGCGAGC-3
0
at the 5
0
-end of the reverse primer must be used.
Purify the resulting PCR product using any available PCR cleanup kit (refer to
Doherty
et al.
, 2010
, e.g. primers).
2.
Digest pYG1 with
Asc
I for 2 h at 37
C to linearise.
3.
Incubate 0.3 pmol of insert with T4 DNA polymerase (1 unit; NEB) and 2.5 mM
dTTP for 30 min at room temperature. Incubate 0.3 pmol of linearised vector
with T4 DNA polymerase (1 unit; NEB) and 2.5 mM dATP for 30 min at room
temperature.
4.
Mix vector and insert in a ratio of
m
1:3 for 10 min at room temperature,
transform into
E
.
coli
and screen as above.
2.1.3
Transforming B. subtilis168
Solutions
10
PC
21.4 g
K
2
HPO
4
12 g
KH
2
PO
4
2g
Na
3
Citrate
5H
2
O
1.7 g
Na
3
Citrate
2H
2
O
Make up to 1 L with dH
2
O and autoclave
MD medium
5mL
10
PC
2.5 mL
40% glucose (w/v)
1.25 mL
trp (2 mg/mL)
0.25 mL
Ferric ammonium citrate (2.2 mg/mL)
2.5 mL
L
-Aspartate (50 mg/mL)
0.15 mL
1 M MgSO
4
