Problem with stable cell line generation?

More Zx Chong's questions See All

Transfected cells died after trypsinization ?

Hi all, I am facing a problem to trypsinize and combine/move the transfected cells into new well . I was planning to pool the transfected cells into a new well to ensure better cell-cell...

11 December 2019 3,243 5 View

Constitutive expression of eGFP in transfected cells?

Hi, I am trying to culture a stable clones using transfected cells. As the clones grow, I noticed the eGFP signal diminishes over time but the cells are still resistant to antibiotics and do not...

11 December 2019 176 14 View

New in lentivirus transduction work?

Hi all, I am new in lentivirus transduction work and have some problem as listed below: 1) Is CMV promoter considered a strong promoter to express the gene I want in the transgenic organism...

09 October 2019 8,592 4 View

Will miRNA transfection affect cell morphology?

Hi Scientist, I am transfecting an oncogenic miRNA into a breast cancer cell line and based on your experience in the mammalian cell line, will such transfection affect the cell morphology?

06 July 2019 7,378 8 View

Suggestion for good plasmid extraction kit?

Hi, Can anyone recommend a plasmid extraction kit that could produce good plasmid yield (>300ng/uL). The plasmid I am using now is a high copy number plasmid and the bacteria I am using is...

06 July 2019 6,820 3 View

MiRNA primer sequence?

Dear all, I have a miRNA primer sequence left by someone else and I would like to know about the sequence of this primer. Is there any method I can use to sequence and know the sequence of this...

05 June 2019 5,879 7 View

Co-transfection of miRNA mimics and inhibitors?

Hi, Is there any paper which reported the co-transfection of miRNA mimics and inhibitors? Thank you.

05 June 2019 9,712 3 View

Use of fluorescence or luciferase approach in checking transfection efficiency?

Hi all, I would like to seek for your opinions on whether fluorescence approach (GFP) or luciferase approach is better in assessing transfection efficiency / success (after miRNA...

03 April 2019 1,383 6 View

Single plasmid that carries both miRNA and reporter gene like GFP/luciferase?

Hi researcher, I wonder any of you have tried to design a single plasmid that carries both miRNA and reporter gene like GFP / luciferase? I am thinking with such plasmid, I will just need to...

03 April 2019 5,144 7 View

Viral or non-viral constructs for transfection?

Hi all, I would like to ask your opinion on whether viral or non-viral construct is better and more superior for transfection involving human breast cancer cell lines, MDA-MB-231? Besides, in...

03 April 2019 6,372 4 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 3 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

Does RNAiMAX transfection reagent work in serum free medium?

I diluted siRNA and RNAiMAX in opti-MEM and added to the cells which they were in the growth medium. Is it a right way? or should I culture cells in the opti-MEM medium for a while and not in...

26 February 2021 10,041 3 View

No amplification data for qPCR on MxPro?

I have set up a qPCR run on MxPro with SYBR green and ROX as a reference, however, when I go to the analysis section the program seems to only collect data for the dissociation curves and not the...

25 February 2021 8,039 1 View

How do I transfect a large plasmid into PC12 cells with nerve growth factor (NGF) receptor?

I have a large 16 kb plasmid, which I need to transfect into PC12 cells. Lipofectamin 2000 didn't work and with GFP alone the transfection rate is very low. I also tried the neon invitrogen...

25 February 2021 6,635 5 View

What is a good reagent for lentiviral plasmid transfection in HeLa cells?

I have been using Lipofectamine 2000 but was hoping there may be a better protocol or one for something like Lipofectamine PLUS reagent.

24 February 2021 1,633 4 View

Uncertianiny in using dry or wet basis in mixing a sediment with fuel oil?

Hello I'm currently working on a topic related to studying the anaerobic biodegradation of TPH in marine sediments. In this work, sediment should be mixed with fuel oil to a concentration of 20 g...

24 February 2021 9,839 3 View

IF troubleshooting: looking at an intracellular protein that localizes to the cell's and how to reduce increase in signal due to cell ruffles?

I am currently trying to stain for PI3KBeta in MDA-MB-231 cells. My current issue is that 1) beta stains all over the cell and 2) there is an increase in signal at the edge of the cell and this...

24 February 2021 8,582 1 View

Is it possible that Cancer cells could survive in heparinized blood for 48 hours?

Hi everyone, I am just wondering if we incubated 200 microliter of whole blood with 50 microliter of cancer cells(1bout 5000cells) do you think that cancer would survive for 48 hours?

23 February 2021 3,840 3 View

Yulia Panina

All of the cells may die due to low transfection efficiency. How many cells do you get transfected?

Zx Chong

Hi Yulia, I am also thinking low transfection efficiency as one of the causes.. My efficiency is very low..

Hi Sebastian, ya, I think I have transfected cells which die after some days.. What inducible system would you suggest?

Thank you both for the suggestions.