Cosmids, phasmids and other advanced vectors - Principles of Gene Manipulation - page 72

Probe
sequence

SP6 promoter

Insert probe

Vector

sequence

Linearize
plasmid

Add SP6 RNA polymerase.
Unlabelled CTP, ATP, GTP.
Labelled UTP

High specific activity
RNA probes

Fig. 5.8 Method for preparing RNA
probes from a cloned DNA molecule
using a phage SP6 promoter and SP6
RNA polymerase.

(a) LITMUS 28 LITMUS 29 LITMUS 38 LITMUS 39

Sna B I
Spe I

Sna B I
Spe I

Sna B I
Spe I

Sna B I
Spe I T7 T7 T7 T7

Bgl II
Nsi I - Ppu 10 I
Bss H II
Bsi W I
Xho I
Eco R I
Pst I
Eco RV
Bam H I
Hin d III
Nco I
Aat II
Age I
Xba I
Avr II
Sac I
Kpn I - Acc 65 I
Stu I

Kpn I - Acc 65 I
Sac I
Avr II
Xba I
Age I
Aat II
Nco I
Hin d III
Bam H I
Eco RV
Pst I
Eco R I
Xho I
BsiW I
Bss H II
Nsi I - Ppu 10 I
Bgl II
Stu I

Apa I - Bsp 120 I
Mfe I
Ngo M I
Kas I
Hin d III
Pst I
Eco RV
Bam H I
Eco R I
Nhe I
Eag I
Mlu I
Bsp E II
Bsr G I
Sph I
Sal I
Stu I

Sal I
Sph I
Bsr G II
Bsp E I
Mlu I
Eag I
Nhe I
Eco R I
Bam H I
Eco RV
Pst I
Hin d III
Kas I
Ngo M I
Mfe I
Apa - Bsp 120 I
Stu I

pUC
origin

M13
origin

pLITMUS TM
2.8 kb

lac Z '

amp r

Afl II T7 Afl II T7 Afl II T7 Afl II T7

Fig. 5.9 Structure and use of the LITMUS vectors for making RNA probes. (a) Structure of the LITMUS vectors showing the
orientation and restriction sites of the four polylinkers.