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Questions related from Michael Sacher
We are following the Zhang lab protocol (Nature Protocols (2013), volume 8, on page 2291 for cloning oligos for gRNA to be used in a CRISPR/Cas9 protocol. We set up the plasmid with BbsI along...
05 May 2019 1,003 14 View
We have used the DQ-BSA assay to examine lysosome activity. We co-localize lysotracker with the DQ-BSA fluorescent signal. In one particular protein knockdown we see normal lysotracker...
08 August 2018 4,591 0 View
How easy is this to do for someone who has never done this before? We have plenty of experience culturing cells but how easy is it to recognize when it has worked? And does anyone have a simple,...
05 May 2018 2,998 4 View
I cannot find a reference for this but assume it ot be the case. If ribosomes are bound to the rough ER during the translocation process, it stands to reason that upon inhibition of translation a...
10 October 2015 703 4 View
We have starved primary fibroblasts in DMEM without any serum. The control fibros seemed to become mobile. They would round up, move, flatten out, repeatedly. In a cell line derived from a patient...
02 February 2015 7,719 3 View
We suspect mycoplasma contamination in one of our cultures since DAPI staining reveals cytoplasmic staining. But one person suggested it was simply mitochondrial. This got me thinking: in all...
11 November 2014 5,810 18 View
We would like to couple a protein of interest to a solid matrix, then pass a lysate over the matrix to see what binds to our protein. What would be a good matrix to use? We have used Affigel from...
12 December 2013 7,750 5 View
We have found that when we boil our sample for 2 minutes in SDS PAGE sample buffer we can detect phosphorylated forms of our protein of interest. But when boiled longer (5 minutes or more), we...
09 September 2013 785 3 View
We have a large yeast strain collection and a plasmid collection. What software are people using to keep their collections organized and searchable? We want to be able to search auxotrophic...
08 August 2013 4,160 4 View
We have expressed human protein in bacterial Arctic Express cells that have two chaperonins in them (Cpn10, Cpn60). Our protein seems to purify along with equal amounts of chaperonin. I assume it...
08 August 2013 5,097 25 View
We have the sequence of an siRNA we have used to knock down a gene of interest. Rather than keep ordering it, we converted it to shRNA to clone into the pU6YH vector. Cloning worked, cells are...
06 June 2013 4,804 4 View
I am cloning a yeast ORF downstream of the ADH1 promoter. For all of our MAMMALIAN work we ensure there is a Kozak sequence upstream of the ATG. But what about yeast? Will I get good expression if...
05 May 2013 849 1 View
Compared to cells like HeLa or 293T, how easy is it to transfect DNA and siRNA into A549 cells? Does it require different/special reagents?
12 December 2012 4,003 9 View
I usually use the Quik Change method using Pfu and complementary oligos. The Phusion (NEB) protocol says to not have overlapping oligos; they should be phosphorylated and then the PCR product is...
09 September 2012 9,952 26 View
