Flower development of Lilium longiflorum - The Lilium information ...

Chapter 4
denaturation, 55 o C for 1 min annealing and 72 o C for 1 min extension, and a final
extension step of 10 min at 72 o C. Five microliters of the PCR reaction were incubated
at 37 o C for 2h with 0.6 µL of restriction buffer and 4 units of HindIII.
The 317-bp PCR product derived from the wild-type AG allele was not
digested by the restriction enzyme whereas the fragment derived from the defective
ag-1 allele was digested into two fragments of 297 and 20 bp. The difference between
the 317-bp and the 297-bp fragment could not be visualised on a non-denaturing
agarose gel. Digested PCR products were denatured and separated on a denaturing
polyacrylamide gel and stained with silver nitrate according to Echt et al. (1996).
Analysis of transgenic plants
Transgenic plants were confirmed by PCR and Southern blots, and the
transgene expression, by northern blots.
Total RNA from Arabidopsis was isolated from flowers using the Rneasy Plant
Mini Kit (Qiagen, GmbH, Hilden, Germany). Ten micrograms of total RNA were
denatured by the glyoxal/DMSO method (Sambrook et al., 1989), and run in a 1.4%
non-denaturing agarose gel prepared with 15 mM sodium phosphate buffer pH 6.5.
Blotting procedures followed the capillary method to transfer nucleic acids to a nylon
membrane (Amersham, Buckinghamshire, UK) with 25 mM phosphate buffer pH 6.5.
Genomic DNA was isolated from leaves by the Dneasy Plant Mini Kit
(Qiagen). Five hundred nanograms of genomic DNA were digested by restriction
enzymes overnight with the appropriate buffer, run on a 0.8% agarose 1X TAE gel and
blotted on nylon membranes by the capillary method, as described by Sambrook et al.
(1989).
Hybridisation procedures were conducted identically for Southern and
northern blots at 60 o C for at least 3h with a previous 1h incubation in the hybridisation
buffer with no probe (10% dextran sulphate, 1% SDS, 1M NaCl and 10 µg mL -1
herring sperm).
A 348-bp fragment corresponding to the specific 3’ end of LLAG1 was used as
a probe and amplified by PCR with the primers 5’-GAT TGC TGA AAA TGA GAG
G-3’ and 5’-AAA GTC ACA AAA TAA TAC AGC-3’ for forward and reverse
annealing at 56 o C. The product was purified by the QIAquick PCR Purification Kit
(Qiagen). About 30 ng of the purified product was taken for radioactive labelling using
[ 32 P] dATP with the RadPrime DNA Labeling System (Invitrogen). The probe was
62