Did anyone ever try using pLVX-TetOne Plasmid with a copGFP as a selection marker instead of Puromycin?

When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...

25 February 2021 1,011 3 View

Why is there no protein expression but antibiotic resistance in transduced A549 cells with pLVX-TetOne-Puro lentivirus?

Hi, I have been trying to transduce A549 and Calu-3 cells using the Lenti-X Tet-One inducible expression system (TakaraBio). My gene of interest is inserted in a pLVX-TetOne-Puro plasmid and has...

15 February 2021 8,462 3 View

Any advice on how to get KO cell lines by santa cruz Crispr-cas9 please?

Hello, I tried several times to obtain two KO cell lines for ERN1 (IRE1 alpha) using a santa cruz crispr cas9 "double nickase". (HNSCC cancer cell lines : PECA-PJ41 and PECA-PJ34). I tried...

07 December 2020 8,141 1 View

Problems with library virus generation?

Dear all I cloned an sgRNA library into a pLenti plasmid backbone (puromycin selection marker) by golden gate ligation and transformation. However I cannot produce any virus from the library...

25 October 2020 3,820 2 View

Antibiotic screening of drug resistance cancer cell line with MDR pumps?

Hi. during our study, we faced with a problem in the screening of EPG RDB 257 cell line which is a resistant cell line due to the expression of efflux pumps such as MDR 1 which actively excrete...

27 August 2020 9,192 1 View

Post transfection contamination ?

i am trying to transfect suspension cell line for that i use lipofectamine 3000 along with reduced serum media without antibiotics (optimem media) 6hr of duration for transfection .After 48 hr i...

19 August 2020 894 3 View

What selection markers to use for the generation of stable HEK293 cell line ?

Dear all, I am trying make stable clones of a HEK293 using an antibiotic selection strategy. However, the parental cell line is already resistant to the five commonly used antibiotics (G418,...

14 August 2020 7,542 2 View

What puromycin concentration would you use for selection in WM3211 melanoma cell line ?

24 May 2020 2,728 4 View

How to create Stble cell line for membrane proteins?

Hi I want to create stable cell lines for membrane proteins of interest in inducible expression system. I wonder if anyone has used pTRE-tight-BI system to express two genes simultaneously in...

12 March 2020 2,832 3 View

What is wrong with discrepancy of sequencing results between gDNA and cDNA after CRISPR/Cas9 gene editing?

Hi everyone, I am trying to use CRISPR/Cas9 to mutate one gene in leukemia cell lines by Lipo3000 (from AGC to AAC). After transfection and puromycin selection of 5 days, the GFP+ of the cells is...

31 December 2019 7,006 3 View