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Questions related to Cloning
The recommended amount of DNA for restriction digestion is 1ug in a 50ul rx I've got around 500-600ng of DNA in a total volume of 35ul. Would I get satisfactory results with this amount of DNA for...
01 February 2021 934 6 View
Hello everybody! so I have a plasmid which has a synthetic intron (IVS) & an internal ribosome entry site (IRES), and I'm thinking to remove them as I'm cloning due to some matters related to...
27 January 2021 7,756 2 View
My secondary antibody ( IgG goat anti rabbit) is immobilised on dextran-coated magnetic nanoparticles, primary antibodies (from rabbit serum) are bound. The primary antibody must maintain its...
20 January 2021 3,953 3 View
I need to fish cDNA of hundreds of genes from cDNA library to clone them in over-expression vectors. I need a reliable human cDNA library. I don't know what are the options, or how should I...
19 January 2021 6,636 0 View
Hello I’ve been trying to clone a gene fragment for some time now, but I'm not getting any results. I did everything according to protocol but I can’t figure out the problem. I amplified my gene...
17 January 2021 6,744 8 View
Hi all, I'm working on transforming E. coli with plasmid in a mixture of linear and circular form. What happened to the linear form once uptake? Do cells ligate them directly? Or cell DNase...
14 January 2021 8,508 4 View
I want to clone have two proteins cloned into pPROexHtb+ (which has one cloning site). I want to express them as a protein complex. My question is would you generally put a stop codon after first...
10 January 2021 8,893 2 View
I have a clone of DNA sequence encoding the full spike protein (S1+S2)
02 January 2021 7,921 1 View
My research is on development of a calibration curve and see effect of river parameters such as flow rate on Ecoi concentration.
31 December 2020 1,210 4 View
hi, I have an simple question related to IRES. Regard with my cloning experiment, I noticed one thing. promoter-gene1-IRES-reporter gene Here is a sequence which I used. In that case, I...
21 December 2020 5,472 2 View
Hi, My experiment is: clone GFP gene into pHG165 plasmid and transform E.coli to express GFP. I did my controls (1 with only cells, 1 with plasmid). But I got low transformation frequency for the...
21 December 2020 5,816 6 View
Hi! I am having problems cloning a 1.6 kb fragment containing 2 different genes and 2X2A peptides into a vector that I already used to clone other fragments (digested with PmeI/BamHI). The vector...
18 December 2020 5,395 3 View
I am trying to do the allelic separation of a ~8 kb region using cloning strategy with pKS plasmid. However, when I analyze the result of the colony PCR sequencing It seems that both alleles were...
15 December 2020 5,934 2 View
I did a gradient PCR from 44C to 66C and got two different bands after gel electrophoresis. I used 10ng plasmid DNA as the template, 0.3mM primers, and the total reaction volume was 20ul. The...
14 December 2020 4,392 21 View
Hi all, I have tried to use Crispr Cas9 ( plasmid px333_cas9_mCherry) to delete some enhancers. I designed sgRNA using Chopchop website to target both sides of an enhancer to induce homologous...
14 December 2020 1,082 3 View
I need to integrate a plasmid into the genome of Pichia pastoris. My plasmid encodes proteins URA3 and TRP1. Therefore I am searching for a P. pastoris strain that is not able to grow on SD-URA...
14 December 2020 9,202 0 View
Need help! I cloned a 1440kb gene of Mycobacterium (Mtb) in pET28a and transformed the BL21 cells. Gene sequence/ORF was checked. Induced the culture at 0.6 O.D at 37°C with 0.1, 0.5 mM and...
13 December 2020 2,710 8 View
Hi, I am working with a plasmid which is known to have very low copy number and when I am isolating it by midiprep alkaline lysis method, I am getting good amount of plasmid i.e., 16.56ng/ul....
12 December 2020 1,480 3 View
Hi there, I am having problems with the insertion of modified DNA (point mutations) into C. glutamicum using the pK19mobsacB integrative system from Schäfer et al. 1994. The same system worked...
10 December 2020 7,759 5 View
I did PCR with Taq, cloned the product and sequenced it. As it is a novel gene, I wanted to confirm the sequence using High Fidelity so I did PCR with High fidelity. Upon sequencing high fidelity...
06 December 2020 991 3 View
I am going to clone gDNA fragment > 10 kb into a pCR TOPO vector. I performed the PCR using KOD PLUS Neo polymerase for 3-steps PCR reaction, I am not able to find the specific size. Several an...
04 December 2020 2,106 5 View
Dear fellow scientists!!! I have been doing cloning lately and have encountered some problems during my experiments, for example, I can get none or very few colonies after transformation into...
03 December 2020 2,344 11 View
Dear Researchers, I cloned the IRF9 coding sequence into plasmid LV073. I used the Gibson assebly (NEBuilder) protocol. I cut the vector in MCS with the enzyme ScaI (blunt ends), amplified my...
27 November 2020 5,166 4 View
I am wholly new to phage display and biopanning. I am looking for a very straightforward explanation as to why suppression of termination of translation is important for phage display.
23 November 2020 8,238 3 View