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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...
06 June 2018 8,818 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...
01 January 2018 9,976 1 View
I have a tab-delimited list with genomic mutations of Drosophila melanogaster mapped to FlyBase r5.50 genome assembly. List contains such fields as gene name, chromosome, strand, genomic...
04 April 2015 2,531 5 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....
02 February 2014 3,433 1 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...
10 October 2013 2,744 17 View
My colleagues and I have an experience of successful cloning into various plasmid vectors. We noticed that it is hard to clone some inserts into one vectors, while the same inserts can be readily...
10 October 2013 4,308 6 View
In yeast genome we have introduced a mutation which replaces transcription factor (TF) binding site to site for restriction endonuclease. The integration marker is URA3. The correctness of marker...
10 October 2013 5,382 6 View
I failed to induce expression of my protein in BL21(pLysS) from pET11c. I obtain the cell culture by growing individual colonies from the plate on LB to OD600 0.5-1. Protein expression is induced...
09 September 2013 8,637 31 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...
09 September 2013 3,697 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,...
09 September 2013 5,926 9 View
We tried to make cycloheximide chase assay in yeasts and found that even 200 mkg/ml concentration of cycloheximide in complete selective media does not stop our protein translation which is very...
12 December 2012 1,955 5 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...
11 November 2012 3,648 8 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...
11 November 2012 4,540 2 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.
11 November 2012 3,595 33 View
Please, suggest a method for estimation (quantification) of DNA repair effeciency (general or specific pathways) in yeasts Saccharomyces cerevisiae.
11 November 2012 7,675 1 View
Can you suggest some software or web-services that perform restriction analysis for DNA sequence containing single nucleotide polymorphism (SNP)? Is there any software that provides comparative...
10 October 2012 304 7 View
Let us imagine that some laboratory developed a method for creating restriction endonucleases that can specifically bind to short DNA sequence and specifically cut it or other determined DNA...
09 September 2012 420 8 View
