The three expression constructs are same except thet the start ATG is placed in three different coding position or in three different open reading frame (ORF) to test translation of potential peptides. The testing construct are introduced into cells by transfection, and the cellular protein extracts are prepared and probed with anti-myc antibody in
Use the link:http://dxys.com/psLHVl lumi package provides an integrated solution for the Illumina microarray data analysis. It includes functions of Illumina BeadStudio (GenomeStudio) data input, quality control, BeadArray-specific variance stabilization, normalization an
Try to have the construct DNA sequenced, make sure that the two enzume sites are correct and located in right positions, and the insert DNA size is right. If cut 1 ug DNA, the cut off 0.3 kb fragment is hard to see on the gel, try cut 10 ug DNA in a final volumes of 200-500 ul. Run 2-2.5 % agarose gel (using low meltng agarose) to purify the
为什么转染结合位点突变的质粒要比野生型质粒的值高两倍多: try to find information about transcription regulation regarding to this DNA fragment mutated at the transcription factor or factors binding site, if gel shift assay or DNase I footprinting shows stronger association of transcription factor or factors with the DNA mutated at DNA binding sites than at the DNA in wild typ
You need to isolate Cresrp Cas 9 /sgRNA transfected cells or cell lines before running T7E1 cleavage assay. The other issue is that PCR amplification of DNAv from sgRNA transfected cells or cell lines would let you run T7E1 easily.Please download the attached file for details.
To make GFP green cells, the transfection efficiency would be above 85% or even high. After transfection, you may perform cell sorting to enrich GFP expression green cells, or you may run cell colony selection to isolate cell clones with high levels of GFP expression.
如果不去背景的话, 我的结果着丝粒处富集的蛋白和染色体臂富集差不多甚至要少一些: you have said that protein A associates with DNA nonspecifically, it indicates that delta t values of no-antibody IPs should be quite same whether DNA at the arms or at CENs. Your results seem to be discrepancy from this assumption.
是不是曝光时间太长: GFP fluorescence excitation spectrum is 488 nm, and its emission 509 nm. As long as GFP protein is in good condition, the blue fluorescence emission will be detected in proportion to amounts of GFP protein.
That is a specific shift formed by the 400 DNA probe and the tested protein. Try to run gel shift using increased amounts of tested protein and constant amounts of DNA probe to optimize the association of protein and the DNA.If it is possible, try to break down the 400 bp DNA probe into 2 or 3 smaller pieces and test each shortened DNA in gel shift