August 3, 2004
Ronna Hertzano
DIG in situ hybridization probes
Pou4f3 probe:
A 484 bp segment of the unique coding sequence of the Pou4f3 gene (NM_138945
– nucleotides 13-496) was cloned into the pGEM T-easy vector (Promega).
For clone#3
Probe
Antisense
Sense
Cut with
SpeI
SalI
NdeI
SacI
BstXI
NsiI
AatII
SacII
Transcribe with
T7
SP6
Gfi1 probe:
A 957 bp segment of the 3' untranslated region of the Gfi1 gene (NM_010278 –
nucleotides 1781-2738) was cloned into the pGEM T-easy vector (Promega).
Probe
Antisense
Sense
Cut with
AatII
SacII
NcoI
SpeI
PstI
SalI
NdeI
SacI
BstXI
NsiI
Transcribe with
SP6
T7
August 3, 2004
Ronna Hertzano
Lhx3 probe: (clone #7)
Probe is based on the 3'UTR of L38248. this 3'UTR is common to Lhx3a and Lhx3b.
isoform specific primers can not be made, as the Lhx3b specific region spans 195bp
and the Lhx3a specific region spans 146bp. The probe is 647bp long and corresponds
to nucleic acids 1428-2074.
Probe
Antisense
Sense
Cut with
ApaI
AatII
NcoI
SacII
SpeI
PstI
SalI
NdeI
SacI
BstXI
NsiI
Transcribe with
SP6
T7
August 3, 2004
Ronna Hertzano