I'm struggling to package a plasmid and want to give phoenix cells a try?
Hi, I'm trying to generate p53/p16 double knockout cell line. I managed to knockout p53 using Crispr. Now, I can't manage to knockdown/out p16. I have tried so far 5 different Crispr targeting...
05 June 2017 7,728 4 View
I'm trying to package pBabe-KRAS, pBabe-beta-catenin and pBabe-PIK3CA retroviral constructs. So far, I have no luck with beta-catenin or PIK3CA although there is no issues with KRAS or empty...
08 September 2016 3,887 3 View
I have two plasmids on the same backbone. Both of them have eGFP. The only difference is that one plasmid has 3x flag before the insert and the other doesn't have. When I transfect HEK293T cells...
31 December 2013 2,577 2 View
I have sub cloned some constructs. I added a flag tag before the insert. I have confirmed the constructs by PCR, diagnostic digestion and sequencing. Now, I'm trying to validate them using western...
09 October 2013 2,118 6 View
I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using...
08 August 2024 4,645 2 View
Hi all, I need to introduce an ARS (autonomously replicating sequence) in my plasmid but I'm not sure which position would be the best. Does anyone have any suggestion? A picture of the plasmid...
05 August 2024 1,573 4 View
I want to introduce a point mutation (change in one nucleotide) into my gene of interest (DNA binding domain) I have designed primers as recommended on the Data sheet of the kit : -Both primers...
05 August 2024 9,059 3 View
I am performing ligation of the plasmid and a target gene. The steps I have taken are: 1. Double digestion of the plasmid and target gene 2. Ligation of the plasmid with the target gene 3....
05 August 2024 2,570 3 View
Dear All, I am trying to transfect a pCDNA3.1 vector containing my gene of interest. The purpose is to figure out the localization of the protein of interest. I have fused the protein with GFP on...
31 July 2024 9,892 4 View
Hello I am trying to create a stable cell line in HEK293 via Lipofectamine 3000 transfection. My plasmid is a CD63-IL10-GFP construct with Puromycin resistance. I am successful with the...
30 July 2024 6,648 1 View
I have been trying to electroporate SKOV3 cells with a large plasmid (11kb) without much success. Any tips?
29 July 2024 3,229 1 View
I created two potential gene expression cassettes (constitutive and inducible) for expression of a mutant PETase gene on PeptiCloud using the version tree feature, which allows users to create...
28 July 2024 7,559 1 View
Please address the best way to drop a plasmid. Background: I have a "bait" plasmid resistant to kanamycin and a "prey" plasmid resistant to carbenicillin. After many rounds of streaking on...
25 July 2024 2,532 3 View
I am currently at a stage in my research where I will be using the pLV6-Bmal-luc plasmid (Addgene #: 68833). However, I am encountering some issues with the isolation of this plasmid and am unable...
23 July 2024 5,296 0 View