I'm working with primary hepatocytes. Nucleofection of large 9.5kb plasmids isn't working but a 4.5kb is. Any experience on RE cutting larger plasmids into pieces then Nucleofecting? Will they be chewed up by cell DNases?
Hi, I have combined two haploid Saccharomyces cerevisiae cultures (opposite mating types) in YPD and want to select only diploids without using solid media. Each haploid has a different selectable...
11 December 2018 2,127 3 View
Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...
01 March 2021 210 1 View
Hello! I'll do a size exclusion chromatography, but I only have an open column, and I'll perform the peptide extraction from yeast, using buffer lysis (sodium phosphate 50 mM/NaCl 30 mM/DNAse and...
01 March 2021 2,215 2 View
Can anyone help me choose primary antibodies for mouse liver hepatocytes and also in conjunction Cd8 cells. Also Cd3 cells. I want to look at hepatocytes and adjacent immune cells in mice with...
01 March 2021 6,953 1 View
I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...
01 March 2021 9,310 4 View
I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...
28 February 2021 5,440 3 View
I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...
28 February 2021 4,949 3 View
Hello, Is it possible to use pUC19 as a transfer vector to be packed in using the second generation viral particles packaging system( pMD2.G; psPAX2 plasmids)? As far as I understand it there is...
28 February 2021 4,868 2 View
When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...
25 February 2021 1,011 3 View
After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify...
25 February 2021 5,712 3 View
I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen...
25 February 2021 6,635 5 View