Biology Reference
In-Depth Information
Avr
II
Kpn
I
Apa
I
Xho
I
Sal
I
Cla
I
Hind
III
Eco
RV
Eco
RI
Pst
I
Avr
II
Kpn
I
Apa
I
Xho
I
Sal
I
Cla
I
Hind
III
Eco
RV
Eco
RI
CCT AGG ATG
GGT ACC GGG CCC
CCC
CTC GAG GTC GAC
GGT
ATC GAT
AAG
CTT
GAT ATC GAA TTC
ATG
CCT AGG ATG
GGT ACC GGG CCC
CCC
P
xyl
CTC GAG GTC GAC
GGT
ATC GAT
AAG
CTT
GAT ATC GAA TTC CTG CAG
ATG
P
xyl
mCherry
gfpmut1
Ori
Ori
pNG621
4.8 kb
pSG1164
4.8 kb
cat
cat
bla
bla
Bam
HI
Apa
I
Xho
I
Sal
I
Cla
I
Hind
III
Eco
RV
Eco
RI
*
Asci
*
CGG ATC CAC
GGG CCC
CCC
CTC GAG GTC GAC
GGT
ATC GAT
AAG
CTT
GAT ATC
GAA TTC
TAG
GAG GGT TCC T
GG CGC GCC
AGC CCA
ATG
P
spac
gfpmut3
yfp
amyE5¢
P
xyl
laci
Ori
pYG1
5.75 kb
pSG1191
7.6 kb
Erm
Spec
Ori
bla
amyE3¢
bla
FIGURE 4.1
Plasmid maps of pSG1164, pNG621, pSG1191 and pYG1. Multiple cloning sites show the
reading frame and all unique restriction enzyme sites are underlines. Stop and start codons
are bold. Asterisks in pYG1 show the nucleotides that the digestion stops at during T4 DNA
polymerase reaction when dTTP is added to the reaction. bla, cat, Erm and Spec, ampicillin,
chloramphenicol, erythromycin and spectinomycin resistance, respectively;
, plasmid
origin of replication; P
xyl
, xylose-inducible promoter; P
spac
, IPTG-inducible promoter.
Plasmids not to scale.
Ori
recombination occurring between plasmid-derived sequences. Furthermore, they
contain compatible antibiotic resistance genes enabling efficient strain construction.
2.1.1
Cloning into pSG- and pNG-based vectors (
Table 4.1
)
1.
PCR amplify the gene of interest using primers that include restriction enzyme sites
chosen from the plasmid's multiple cloning site (MCS). Confirm that these sites are
not present
within
the amplified PCR products. Purify the resulting PCR product
using any available PCR cleanup kit (refer to
Figure 4.1
for restriction sites).
