E washes and elution buffers contained 20 mM and 250 mM of imidazole respectively.Motif GenerationThe 27 clones which tested positive for binding to Mid in an EMSA were entered into MEME to generate a motif [10]. The relevant parameters were set such that every sequence was used once to generate a motif with a length of 6?6 nucleotides. The sequences of primer F and primer R were used as negative sequences. Motifs in. Figure 1B were generated using WebLogo [41]. Sequences for the Tbx20 motif were obtained from MacIndoe et al. [6], while those for Mid were generated from data from Liu et al. [18].Non-Radioactive Electro-mobility Shift AssaysProbes used for EMSAs were generated from pCRII or pCR2.1 clones by PCR amplification of the cloned oligonucleotide using 59-biotin-labelled primer F and primer R (see above for sequence). The PCR product was phenol/chloroform extracted and ethanol precipitated in the presence of glycogen. The T-site probe corresponding to the Bs.p Tunicamycin site palindrome (AATTTCACACCTAGGTGTGAAATT) was obtained as a self-complimentary primer with 59 biotin labels. Tbx20.MacIndoe (HIF-2��-IN-1 chemical information GGAGGTGTGAGGCGA and TCGCCTCACACCTCC), mid.Liu (GGAAGTAGGTCAAG and CTTGACCTACTTCC), mid.Najand (CAAGGTGTCAAGGCG and CGCCTTGACACCTTG), mid.Najand Region 1 (CACCCCCCCAAGGCG and CGCCTTGGGGGGGTG) and mid.Najand Region 2 (CAAGGTGTCAAGGAA and TTCCTTGACACCTTG) were ordered as 59 biotin-labelled primers and annealed to their complement in 1X Taq polymerase buffer. A 10 ml reaction containing 15 fmol of each biotin-labeled probe, 375 ng of purified 6x-His MidTbx and binding buffer (20 mM HEPES pH 7.9, 100 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 20 glycerol, 0.1 nonidet P40, 0.5 mM DTT, 10 mg/ml BSA, 8 ng/ ml poly(dI-dC)Npoly(dI-dC)) was assembled and incubated at room temperature for 1 hr. The sample was loaded onto a 8610 cm 5 polyacrylamide, 2.5 glycerol gel in 0.5X TAE running buffer, pre-run at 85V for 1 hour. mid.Najand oligonucleotides were run on a 10 polyacrylamide gel containing 10 glycerol. Once loaded the sample was run for 5 min at 120 V and 1 hour at 85 V for 5 gels, and 2 hours for 10 gels. The oligonucleotide was transferred onto a Hybond-N+ nylon membrane (Amersham) at 85 V for 30 min in 0.5X TAE. Following transfer, the oligonucleotides were cross-linked to the membrane using a transilluminator and visualized using the chemiluminescent nucleic acid detection module (Pierce) according to the manufacturer’s directions. All probes were run a minimum of 2 times to confirm that MidTbx is able to bind and retard their mobility. Probes that showed no binding were run 3? times to ensure a negative result.Site SelectionSite selection was carried out essentially as described [28] with modifications such that it could be carried out non-radioactively. Oligonucleotide R76: CAGGTCAGTTCAGCGGATCCTGTCG(N26)GAGGCGAATTCAGTGCAACTGCAGC, which consists of a 26 nucleotide random core flanked by primer sequences was rendered double stranded using Taq DNA polymerase and primer F (GCTGCAGTTGCACTGAATTCGCCTC), and was purified using High Pure PCR Cleanup Micro Kit (Roche). A 25 ml reaction containing 0.4 ng of purified, double-stranded primer F, 550 ng of purified 6x-His MidTbx protein and binding buffer (20 mM HEPES pH 7.9, 100 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 20 glycerol, 0.1 Nonidet P40, 0.5 mM DTT, 10 mg/ml BSA, 8 ng/ml poly(dI-dC)Npoly(dI-dC)) was assembled and incubated at room temperature for 1 hr. The reaction was added to 10 ml of 5 NiNTA magnetic beads (Qiage.E washes and elution buffers contained 20 mM and 250 mM of imidazole respectively.Motif GenerationThe 27 clones which tested positive for binding to Mid in an EMSA were entered into MEME to generate a motif [10]. The relevant parameters were set such that every sequence was used once to generate a motif with a length of 6?6 nucleotides. The sequences of primer F and primer R were used as negative sequences. Motifs in. Figure 1B were generated using WebLogo [41]. Sequences for the Tbx20 motif were obtained from MacIndoe et al. [6], while those for Mid were generated from data from Liu et al. [18].Non-Radioactive Electro-mobility Shift AssaysProbes used for EMSAs were generated from pCRII or pCR2.1 clones by PCR amplification of the cloned oligonucleotide using 59-biotin-labelled primer F and primer R (see above for sequence). The PCR product was phenol/chloroform extracted and ethanol precipitated in the presence of glycogen. The T-site probe corresponding to the Bs.p palindrome (AATTTCACACCTAGGTGTGAAATT) was obtained as a self-complimentary primer with 59 biotin labels. Tbx20.MacIndoe (GGAGGTGTGAGGCGA and TCGCCTCACACCTCC), mid.Liu (GGAAGTAGGTCAAG and CTTGACCTACTTCC), mid.Najand (CAAGGTGTCAAGGCG and CGCCTTGACACCTTG), mid.Najand Region 1 (CACCCCCCCAAGGCG and CGCCTTGGGGGGGTG) and mid.Najand Region 2 (CAAGGTGTCAAGGAA and TTCCTTGACACCTTG) were ordered as 59 biotin-labelled primers and annealed to their complement in 1X Taq polymerase buffer. A 10 ml reaction containing 15 fmol of each biotin-labeled probe, 375 ng of purified 6x-His MidTbx and binding buffer (20 mM HEPES pH 7.9, 100 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 20 glycerol, 0.1 nonidet P40, 0.5 mM DTT, 10 mg/ml BSA, 8 ng/ ml poly(dI-dC)Npoly(dI-dC)) was assembled and incubated at room temperature for 1 hr. The sample was loaded onto a 8610 cm 5 polyacrylamide, 2.5 glycerol gel in 0.5X TAE running buffer, pre-run at 85V for 1 hour. mid.Najand oligonucleotides were run on a 10 polyacrylamide gel containing 10 glycerol. Once loaded the sample was run for 5 min at 120 V and 1 hour at 85 V for 5 gels, and 2 hours for 10 gels. The oligonucleotide was transferred onto a Hybond-N+ nylon membrane (Amersham) at 85 V for 30 min in 0.5X TAE. Following transfer, the oligonucleotides were cross-linked to the membrane using a transilluminator and visualized using the chemiluminescent nucleic acid detection module (Pierce) according to the manufacturer’s directions. All probes were run a minimum of 2 times to confirm that MidTbx is able to bind and retard their mobility. Probes that showed no binding were run 3? times to ensure a negative result.Site SelectionSite selection was carried out essentially as described [28] with modifications such that it could be carried out non-radioactively. Oligonucleotide R76: CAGGTCAGTTCAGCGGATCCTGTCG(N26)GAGGCGAATTCAGTGCAACTGCAGC, which consists of a 26 nucleotide random core flanked by primer sequences was rendered double stranded using Taq DNA polymerase and primer F (GCTGCAGTTGCACTGAATTCGCCTC), and was purified using High Pure PCR Cleanup Micro Kit (Roche). A 25 ml reaction containing 0.4 ng of purified, double-stranded primer F, 550 ng of purified 6x-His MidTbx protein and binding buffer (20 mM HEPES pH 7.9, 100 mM KCl, 0.2 mM EDTA, 0.2 mM EGTA, 20 glycerol, 0.1 Nonidet P40, 0.5 mM DTT, 10 mg/ml BSA, 8 ng/ml poly(dI-dC)Npoly(dI-dC)) was assembled and incubated at room temperature for 1 hr. The reaction was added to 10 ml of 5 NiNTA magnetic beads (Qiage.

E washes and elution buffers contained 20 mM and 250 mM of imidazole

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