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Materials and Methods
Analysis of Primary Cases.
Total genomic DNA was isolated from sections of the lymph nodes using the QIAamp kit. Single H/RS cells were micromanipulated from 5-μm histological sections stained for CD30, and stored in 20 μl of 1× Expand PCR buffer (Boehringer Mannheim) at −20°C. Thawed samples were incubated with 0.25 mg/ml proteinase K (Boehringer Mannheim) for 2 h at 50°C and then 8 min at 96°C. For amplification of exons 1 and 2, samples were adjusted to 50-μl reaction mixes supplemented with 2.5 mM MgCl2, 4% DMSO, 50 nM each of primers IκB1b (5′-TGGTCTGACTGGCTTGGAAATTC-3′) and IκB1c (5′-CATCGCTGGTCCCCCGGCTC-3′), and 16.6 nM of primers IκB2a (5′-CGAAGTCCCCGGTTGCATAAGG-3′) and IκB2c (5′-GGATCTGGGGTGACTCTGCTAC-3′). After incubation for 5 min at 95°C, 35 cycles of 50 s at 95°C, 30 s at 65°C, and 90 s at 72°C were followed by 10 min at 72°C. For amplification of exons 3–6, samples were supplemented with 2 mM MgCl2, 50 nM each of primers IκB3a (5′-CCTGTCTAGGAGGAGCAGCAC-3′), IκB3d (5′-TAGGAGTTTAAGCTCTTGCCTGGA-3′), IκB4b (5′-AAAGAATAGGTGAAAGGAGTGAGG-3′), and IκB4c (5′-ATAAGCACGAGGAGCCTGACTCA-3′), and 16.6 nM each of primers IκB5a (5′-AGCAGAAATTCCAAATGCAGCCAT-3′), IκB5d (5′-GGAGCAGCTCTAGGGGCCTG-3′), IκB6a (5′-GAGTTATTTCCAGTAGTGGCCTC-3′), and IκB6d (5′-GGGGTCAGTCACTCGAAGCAC-3′). PCR was as above, except that the annealing temperature was 63°C. Seminested amplification of the individual exons was as follows: for exon 1, the Expand™ PCR system was supplemented with 2 mM MgCl2, 4% DMSO, and 125 nM each of primers IκB1b and IκB1r (5′-GCGTCCCGCCCTCCCGACGA-3′); for exon 2, the Expand™ PCR system was supplemented with 2 mM MgCl2 and 125 nM each of primers IκB2b (5′-AGTACAGGTCGTTCCGAGCTGG-3′) and IκB2c; exons 3–6 were amplified using standard Taq DNA polymerase in 2 mM MgCl2; 125 nM each of primers IκB3b (5′-AACCAGGAGACACGGGTTGAGG-3′) and IκB3d, IκB4b2 (5′-GAGGGTTGAAACAGGTGGTTAT-3′) and IκB4c, IκB5a and IκB5c (5′-GGAGGGTGAAGGGAATGGCAC-3′), and IκB6b (5′-CCC-ATCCCGGTAGCTTGGCAG-3′) and IκB6d were included, respectively. After 5 min at 95°C, 45 cycles of 50 s at 95°C, 30 s at 65°C, and 90 s at 72°C were followed by 10 min at 72°C. Amplification of products spanning several exons was performed accordingly. The same conditions were also used for amplification of the IκBα exons from total genomic DNA of cell populations and cell lines. Products were gel-purified and sequenced directly using the ABI BigDye system.
For amplification of exons 1 and 4 from aliquoted DNA, single H/RS cells were incubated with 0.25 mg/ml proteinase K (Boehringer Mannheim) in 120 μl 1× PCR buffer containing 1 ng/μl 5S rRNA and incubated at 50°C for 14 h. After gentle mixing by 10 pipetting steps, 20-μl aliquots of the reactions were heated to 96°C for 8 min and subsequently adjusted to 50-μl Expand PCR reactions supplemented with 2 mM MgCl2, 4% DMSO, 125 nM each of primers IκB1s (5′-CTGAAGAAGGAGCGGCTACTG-3′), IκB1c, and IκB4b, and 62.5 nM of primer IκB5c. After incubation for 5 min at 95°C, 35 cycles of 50 s at 95°C, 30 s at 63°C, and 90 s at 72°C were followed by 10 min at 72°C. Seminested amplification of exon 1 (the Expand™ system was supplemented with 2 mM MgCl2, 4% DMSO, and 125 nM each of primers IκB1s and IκB1r) and exon 4 (standard Taq DNA polymerase was used in 2 mM MgCl2 and 125 nM each of primers IκB4b2 and IκB5c) was essentially as above.

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