12 Questions 36 Answers 0 Followers
Questions related from Dmitry S Karpov
As I know there are particular DNA sequences that mediate efficient import of plasmids in into the nucleus, for example, cPPT sequence of the lentiviral vectors (one of its functions is...
04 June 2018 3,288 2 View
I am working with highly mutagenized Bacilli species and wish to measure expression level of my favorite genes by real-time PCR. As I know, there are no reliable internal reference genes for...
14 January 2018 1,726 0 View
I am trying to delete a gene of interest in D.hansenii. There is a protocol for transformation of this yeast by electroporation, but for me it is more convenient to use chemical transformation....
24 February 2014 6,999 1 View
We will try to induce sporulation of a yeast strain transformed with a low copy plasmid. Is it possible, that plasmid can be lost during sporulation on media containing yeast extract? Could you...
16 October 2013 7,930 7 View
We have cloned a yeast gene for a large protein (ca mass about 93 kDa) together with its native promoter into plasmid where our protein is C-terminally tagged with 3xHA epitope. Insert together...
09 October 2013 9,312 16 View
I try to display an enzyme on the surface of E.coli cells. To do this I have made a chimeric protein consisting of signal peptide (SP) - enzyme (E) - outer membrane protein of E.coli (MP). The...
23 September 2013 5,085 7 View
Standard complex media like LB that is routinely used for cultivation of E.coli and performing various experiments including protein expression has several serious limitations. For example,...
18 September 2013 7,557 8 View
I am unable to clone in E.coli a gene for restriction endonuclease that is supposed to be used in an eukaryotic organism. The reason is that the strain of E.coli (DH5alpha) has no protection from...
23 June 2013 8,220 9 View
To measure protein half-lives in cultured mammalian cells (HEK293), we used cycloheximide at a concentration of 100 mkg/ml. The control protein which is GFP fused to the C-terminal degron of...
20 February 2013 9,102 7 View
Can anyone suggest a fluorescent DNA-binding dye that gives the highest signal to background ratio in comet assay. Also can anyone suggest a good software that is suitable for yeast comets' analysis.
19 November 2012 1,526 35 View
Can anyone suggest a reliable protocol for purification of nuclei from yeast cells (Saccharomyces cerevisiae) and preparation of cytosolic fraction for Western-blot analysis? What proteins can be...
15 November 2012 2,542 2 View
Monomeric forms of fluorescent proteins like GFP may aggregate when they are fused to other proteins which are expressed under high levels from strong promoters. One of the solutions of this...
15 November 2012 4,775 7 View
