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Questions related to Cloning
Hello, Does any one faced transgene silencing after transfection? I have been trying to get good expression from a recombinant antibody molecules cloned in pUC based vector and transfected in CHO...
20 February 2020 3,034 7 View
I am trying to clone a 1.3 kb fragment using the pJet system. It is a sticky end cloning protocol and I have tried different ratios (from 3:1 to 8:1). I have used DH5a and Top10 cells with...
19 February 2020 9,286 3 View
Hi, I'm currently working with the Flp-In T-REx system to get isogenic stably transfected cells expressing my GOI (gene of interest, of about 1.4 Kb). I have generated a Flp-In Cell Line and since...
18 February 2020 5,707 2 View
We performed a CRISPR to knock out XPF in a RPE1 cell line and want to "pre-validate" clones through UV-C hypersensitivity (254 nm lamp). Which dose would you suggest to use to maximize...
18 February 2020 8,116 3 View
The commonly used CAG promoter is usually labeled as three parts, take pCAGGS-mCherry (https://www.addgene.org/41583/) for example. CMV enhancer + chicken beta-actin promoter + chimeric...
14 February 2020 7,312 2 View
I am trying to tag moxCerulean3 to the N-terminus of a gene of interest. In other words, I'd like to place this reporter on the first exon of the gene right after the start codon. I've been using...
10 February 2020 7,821 1 View
I was wondering if we could skip one step in cloning process from sequencing vector to expression vector? Meaning that start cloning the gene directly into expression vector such as pET 28b and do...
05 February 2020 9,857 2 View
In order to align sequences i use Clone Manager, however, when I try to align 4 or more sequences, I do have problems with the alignment...Some of the sequences which should align 100% with the...
29 January 2020 9,630 2 View
We are conducting FIV study and would like to stain feline PBMC's for CD25. One vendor calls the 3G10 clone "pan mammalian" but doesn't include feline in the list of known cross reactive species....
28 January 2020 9,059 1 View
Hello everyone We've been struggling with our project due to low efficiency rates + check the insert is OK and not in reverse + PolTaq did not introduce a new mutation... and it has come to our...
24 January 2020 6,149 3 View
Research enigma! Cloning in lactobacillus! After transformation I got complete white colonies but after sub culturing the same , It is starting to see red colonies.No idea from where I got those...
21 January 2020 6,449 3 View
Hi I’m bit confused As I done ligation but I was trying to check if my ligation is successful or not I run my sample on gel but I didn’t found any band as I was having 6 sample .I’m trying to...
20 January 2020 1,416 5 View
I want to clone a gene in pPICZ alpha vector. Is there need to Pichia Kozak consensus (which is (G/A)NNATGG) need to optimize?
19 January 2020 2,885 3 View
Hi all, I am writing manuscript for cloning research, I want to ask if there's any software that allow us to generate nucleotide and amino acid sequence in one same figure, I have been doing using...
16 January 2020 6,424 3 View
Hi, Im working on a gram negative bacteria which are not a model organism. Im looking for strong promoters for expressing both the guide RNA and the CAS9 protein for CRISPR. Any recommendations?
16 January 2020 9,538 1 View
Hello, I am trying to express a fusion protein using a pRSET A vector in C41(DE3) E. coli strains. I have tried many ways and I am trying to see if my sequence is correct. My question is if it is...
14 January 2020 8,020 2 View
I'am new in seamless cloning, I don't know how it works. Can someone help or give a doc where I can learn about that? i'm talking precisely about abclonal seamless cloning.
09 January 2020 3,049 3 View
In short, I am trying to get a clone of 1664 bp insert in a mammalian expression vector (Size of vector is 6250 bp) by using NEBuilder HiFi DNA assembly Cloning kit with commercial Comp cells...
07 January 2020 616 3 View
I need to know the sequence of amino acids for some genes, and the way to detect them
26 December 2019 3,243 5 View
Good evening evryone. I am actually working on dsRNA mycoviruses; but very recently, de HiSeq on my sample revealed the presence of an ssRNA virus. Therefore, i would like anyone who can give me...
23 December 2019 4,684 1 View
I knocked out a gene which is expressed in skeletal muscle, one is hsp40 and another one is a co-chaperone of hsp90a1. Previous study showed that hsp90a1 is specially expressed in skeletal muscle,...
13 December 2019 5,265 4 View
I am transforming 3 different vectors in E coli competent bacteria: cDNA3.1, Pe16b and Pe28a+. I used the same protocol (heat shock) for transformation and plating ( agr + appropriate...
12 December 2019 3,352 2 View
Hi all, I have been trying to do some cloning recently. When I am doing the ligation, I have 1 control: No insert control. In the no insert control, I did not put insert into the vector, which is...
09 December 2019 545 3 View
We have purchased an expression vector from DNASU (pANT7_cGST). The expression vector has a human protein cDNA as insert. we have made multiple attempts to express the protein in different con of...
09 December 2019 3,051 8 View