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Antisense Treatment: Two commercially available Tbx2oligonucleotides were used to block the sense Tbx2(obtained from Oligos Etc.). One antisense is complentary to the portion of mRNA coding for the N-Terminus of the TBX2 protein called N- Terminus. The other antisense is complentary to the portion of mRNA coding for the Box (DNA binding domain) region of the TBX2 protein, called Box antisense. The three experimental culture conditions were as follows: 1) N-Terminus antisense oligonucleotide; 2) Box antisense oligonucleotide; 3) N-Terminus antisense oligonucleotide and Box antisense oligonucleotide combined. The oligonucleotides were introduced as a 200nM concentration in 0.66% Lipofectin in OptiMEM (Life Technologies) as previously described (Oligos Etc. protocol). The incubation in antisense occurred at 37oC for four hours. The control condition contained 200 nM of sense Tbx2in 0.66% Lipofectin in OptiMEM.

RT-PCR:The RNA was extracted from the explants by homogenizing the cultured explants in phenolguanidium and precipitated as previously described (Cebra- Thomas protocol). Reverse Transcriptase was added to the extracted RNA to form cDNA. PCR experiments were run for 35 cycles at an annealing temperature of 65oC in the Minicycler apparatus using the synthesized cDNA in the presence of buffer, deoxyribonucleotide phosphates (dNTPs), and T. aquaticus polymerase in a final volume of 50ml. Three cDNAs were run in each of three different primers as follows (cDNA/Primer):

©Cebra-Thomas, 2000

Last Modified: 05 May 2000

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