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Questions related to Cloning
I am doing a mutation in a 80% approx. GC content region of my cDNA in a plasmid. I am using the Agilent QuikChange XL II mutagenesis kit. I've used primers designed by the Agilent's software...
17 April 2021 2,153 3 View
in vito studies
15 April 2021 9,960 1 View
Hello everyone, I am doing yeast transformation? I should grow the starting yeast in 5-FoA/YPD to select URA(-) strains. Why do I need to do this step? Is it because URA(+) strains habouring URA...
13 April 2021 3,272 3 View
To give some background, I was instructed by my supervisor to analyse the relationship between the bacteria using the measurements listed in the title and to see whether they were in the same...
11 April 2021 5,378 2 View
I am doing starch-iodine assay for measure the enzyme activity. I use starch as substrate. I add the soluble starch to the form of a paste and slowly add it into the hot water. When I start...
03 April 2021 1,076 8 View
I have been trying to clone a sequence into a single site in the vector. I was able to make about 10 clones so far, but all of them had the insert in the wrong orientation. I would expect 50% of...
23 March 2021 3,318 18 View
is there any trouble that would result from incubating Quick CIP for 1 hour. NEB recommendation is to do it for 10 min only, isn't that a bit too short or has anyone tried it and it works just...
11 March 2021 708 6 View
Is it possible to induce site-directed substitution mutation by quick-change method on linear dsDNA? or it has to be cloned in some vector? If yes, should it be treated with the Dpn1 enzyme...
04 March 2021 710 4 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...
02 March 2021 502 1 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...
01 March 2021 5,708 3 View
I am facing difficulties in cloning a 1kb gene into a vector (pJIT163). I have my gene on interest (GOI) in pUC57 and want to clone in pJIT163 using SalI and BamHI restriction sites. I am getting...
26 February 2021 3,435 7 View
The guide RNA is 5'-17n's-GGG-3'. There is no other PAM adjacent to the 3' end that is not part of the gRNA. This is intended to be a simple double-stranded cut to knock out the gene.
21 February 2021 550 1 View
I have a Crispr/Cas9 edited bulk population and I can briefly check the indel ratios in the bulk population using Sanger sequencing. After cloning single cells, I wanted to check the indel types...
19 February 2021 8,366 6 View
Hi, I am stuck with a cloning experiment: The insert & backbone(bb) share one compatible end at 3' end(NotI) and one incompatible end at 5' end (XbaI for insert; HindIII for backbone). So i...
17 February 2021 1,837 3 View
Hello. I am trying to make competent cells and the transformation buffer in my protocol requires MnCl2. I don't have it, but I do have MgCl2 and am wondering if it will work the same. The...
17 February 2021 9,219 3 View
Hello! I am trying to generate a construct in this format: seqA - seqB - linker - seqC - seqB into pSF-CMV-Ub-Puro-Ascl plasmid 0.5kb 2kb 66 0.25kb 2kb I have tried many...
16 February 2021 7,289 3 View
Hello all, I am interested in deleting a highly expressed non-coding RNA located on a Listeria monocytogenes plasmid. Currently, there are not any optimized gene deletion methods for Listeria...
10 February 2021 5,147 1 View
It seems Creative Biolabs has everything and so many versions of antibodies not owned or sold by any other company. How reliable are their products? I am looking for a clone where potentially...
09 February 2021 2,327 3 View
Dear all, I´m planing to transduce white pre-adipocytes with a lentiviral constructs for inducible shRNA-mediated knockdown of certain target genes. Since my target genes are strongly involved in...
09 February 2021 9,262 4 View
Hello Everyone, We are inexperienced in culture of MSCs trying to grow a sample from BM of a mare. It is the second time that after preparation of new medium (DMEM-F12 with high glucose - 10% Hy...
08 February 2021 10,187 7 View
Hi everyone, I need to clone in pGEM-T vector the result of amplifying a metagenomic library by PCR with a high fidelity polymerase but I only want to clone the fragments of an specific size...
05 February 2021 6,239 8 View
I am trying to clone a pGreenPuro vector (size 8kb) into stbl3 cells but eventually there is no colony on the plates. I plate them 37 ’C overnight on the LB-agar plates. Any solution?
04 February 2021 1,356 5 View
I measured the DNA concentration using Nanodrop and made 10 μl total digest solution. I was wondering what would be the problem that I don't even get band for the uncut control sample?!
04 February 2021 741 6 View
Hi all, I want to express several genes in S.aureus by using the pRMC2 expression vector. I want to try to subclone some genes already cloned into pRMC2 (and their own tetO promoter) and put them...
02 February 2021 5,268 3 View