The aims of this work were to: i) purify GST-fusion protein from bacterial cell extracts of Escherichia coli;ii) quantify the protein by SDS PAGE and Bradford assay;iii) determine protein-DNA interaction of the purifi...The aims of this work were to: i) purify GST-fusion protein from bacterial cell extracts of Escherichia coli;ii) quantify the protein by SDS PAGE and Bradford assay;iii) determine protein-DNA interaction of the purified protein by Electrophoretic Mobility Shift Assay. Bacterial culture prepared by inoculation of a single E. coli colony that had a GST fusion protein (gst: six-X10 hd) constructed by ligation of the six-7-hd (X10) sequence into the BamHI and EcoRI sites of the vector pGEX-2T, grown overnight, was sonicated using Cole-Palmer Ultrasonic Homogenizer. Fusion protein was eluted from the beads with Tris-glutathione buffer (50 mM Tris [pH 8.1], 20 mM glutathione), which contained reduced Glutathione. SDS-PAGE was used to calculate the extracted bound protein. Total protein quantification was then estimated by the Bradford assay. Bovine Serum Albumin (BSA) absorbance values were used to plot the standard curve used to calculate the concentrations of the sample proteins. Nylon membrane was used for the electrophoretic transfer;membrane was cross linked and detected by Pierce’s Chemiluminescent Nucleic Acid Detection module. Results showed that X10 gave a strong band shift observed in Lanes 6 and 7 for both 200 ng and 400 ng elute 1 samples;however, there was no shift in the bands for the wild-type, positive control. The concentration of the elute 1 was obtained by the Bradford assay as 242.52 ng/μl and that of elute 2 was 106.30 ng/μl. Similarly, the result obtained by gel analysis was 300 ng/μl (0.3 μg/μl) and 150 ng/μl (0.15 μg/μl) for elutes 1 and 2 respectively.展开更多
Morphological descriptors and Random Amplification of Polymorphic DNA (RAPD) technique were used to assess the genetic variation among and within forty Pennesitum glaucum accessions from Sudan. Accessions were collect...Morphological descriptors and Random Amplification of Polymorphic DNA (RAPD) technique were used to assess the genetic variation among and within forty Pennesitum glaucum accessions from Sudan. Accessions were collected from 30 villages representing Darfur, North Kordofan, South Kordofan, and Blue Nile states. 64 amplified fragments were distinguished using ten primers.? 63 bands were polymorphic among the forty accessions with an average of 6.3 polymorphic bands per primer. Low level of genetic similarity was observed (4% - 43%). The PhiPT (analogue of FST fixation index) value for genetic variability obtained over the four regions was 0.169 with high significance (P = 0.01). AMOVA analysis showed higher variance components within regions (80%) than among regions (20%). The two dendrograms obtained by Random Amplification of Polymorphic DNA (RAPD) data;and morphological data based on 26 descriptors did not fit together. PCA (Principal coordinate’s analysis) showed geographic structuring of pearl millet according to its growing regions in Sudan.展开更多
文摘The aims of this work were to: i) purify GST-fusion protein from bacterial cell extracts of Escherichia coli;ii) quantify the protein by SDS PAGE and Bradford assay;iii) determine protein-DNA interaction of the purified protein by Electrophoretic Mobility Shift Assay. Bacterial culture prepared by inoculation of a single E. coli colony that had a GST fusion protein (gst: six-X10 hd) constructed by ligation of the six-7-hd (X10) sequence into the BamHI and EcoRI sites of the vector pGEX-2T, grown overnight, was sonicated using Cole-Palmer Ultrasonic Homogenizer. Fusion protein was eluted from the beads with Tris-glutathione buffer (50 mM Tris [pH 8.1], 20 mM glutathione), which contained reduced Glutathione. SDS-PAGE was used to calculate the extracted bound protein. Total protein quantification was then estimated by the Bradford assay. Bovine Serum Albumin (BSA) absorbance values were used to plot the standard curve used to calculate the concentrations of the sample proteins. Nylon membrane was used for the electrophoretic transfer;membrane was cross linked and detected by Pierce’s Chemiluminescent Nucleic Acid Detection module. Results showed that X10 gave a strong band shift observed in Lanes 6 and 7 for both 200 ng and 400 ng elute 1 samples;however, there was no shift in the bands for the wild-type, positive control. The concentration of the elute 1 was obtained by the Bradford assay as 242.52 ng/μl and that of elute 2 was 106.30 ng/μl. Similarly, the result obtained by gel analysis was 300 ng/μl (0.3 μg/μl) and 150 ng/μl (0.15 μg/μl) for elutes 1 and 2 respectively.
文摘Morphological descriptors and Random Amplification of Polymorphic DNA (RAPD) technique were used to assess the genetic variation among and within forty Pennesitum glaucum accessions from Sudan. Accessions were collected from 30 villages representing Darfur, North Kordofan, South Kordofan, and Blue Nile states. 64 amplified fragments were distinguished using ten primers.? 63 bands were polymorphic among the forty accessions with an average of 6.3 polymorphic bands per primer. Low level of genetic similarity was observed (4% - 43%). The PhiPT (analogue of FST fixation index) value for genetic variability obtained over the four regions was 0.169 with high significance (P = 0.01). AMOVA analysis showed higher variance components within regions (80%) than among regions (20%). The two dendrograms obtained by Random Amplification of Polymorphic DNA (RAPD) data;and morphological data based on 26 descriptors did not fit together. PCA (Principal coordinate’s analysis) showed geographic structuring of pearl millet according to its growing regions in Sudan.
