Supplementary
Information
Figure
1. Inverse PCR
(IPCR)
Supplementary
Table 1. Primer
sequences used for inverse PCR reactions and
sequencing
Supplementary
Table 2. Inverse
PCR Reactions and Cycling Conditions
Figure
1. Inverse PCR method to map
mini-Tn5-lux transposon insertion sites.
Genomic DNA was isolated (1) and digested
with NarI or SstII (2). Linear genomic
fragments were circularized by ligation
with T4 DNA Ligase (3). Ligation product
was used as template for an inverse
PCR (IPCR) reaction using outward facing
transposon-specific primers (4). IPCR
products were purified for sequencing
with a nested transposon-specific primer. |
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Supplementary Table
1. Primer sequences used for inverse
PCR reactions and sequencing
| NarI–digested
gDNA |
Primer
sequence 5’ – 3’ |
| IPCR
primer 1 (Tn5-NarI) |
CATTCAGGTCGAGGTGGCCCG |
| IPCR
primer 2 (Tn5-out2) |
CAGAACATATCCATCGCGTCCGCC |
| Sequencing
primer (Tn5-out3) |
CGGTTTACAAGCATAAAGCTTGC |
| |
|
| SstII-digested
gDNA |
|
| IPCR
primer 1 (Tn5-SstII) |
GTCAAAAGGACGATTTCGGTTTGG |
| IPCR
primer 2 (Tn5-out) |
GATCCCCGGGTACCGAGCTCGAATTC |
| Sequencing
primer (Tn5-out-seq) |
CCGGGTACCGAGCTCGAATTCG |
| |
|
| SphI–digested
gDNA |
|
| IPCR
primer 1 (Tn5-SphI) |
GACATGCGGATGTTATTGTCGCTTGGG |
| IPCR
primer 2 (Tn5-out) |
GATCCCCGGGTACCGAGCTCGAATTC |
| Sequencing
primer (Tn5-out-seq) |
CCGGGTACCGAGCTCGAATTCG |
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Supplementary
Table 2. Inverse PCR Reactions and
Cycling Conditions
|
Reagent |
Volume
(ml) |
Step |
PCR
Cycling Conditions |
|
Template
(Ligation reaction) |
2.5 |
1 |
95°C for
3 min |
|
10
mM dNTPs
(2.5 mM each) |
2 |
2 |
95°C for
30 sec |
|
10
mM Primer
1 |
0.5 |
3 |
65°C for
1 min (as
low as 58°C) |
|
10
mM Primer
2 |
0.53 |
4 |
72°C for
3 min |
|
10
X Invitrogen Buffer |
2.5 |
5 |
Go
to Step 2, 4X |
|
50
mM Invitrogen
MgCl2 |
1 |
6 |
95°C for
30 sec |
|
H20 |
15.9 |
7 |
60°C for
1 min (as
low as 58°C) |
|
Invitrogen Taq
(5 U/ul) |
0.125 |
8 |
72°C for
3 min |
|
Final
Volume |
25 |
9 |
Go
to Step 6, 29X |
|
|
10 |
4°C forever |
|
