Is it very hard to transfect Hs578T cell ?

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Toxicity of Lipofectamine 3000?

Dear all, I have been struggling with transfection in Hs578t with my pure plasmid ( maxi prep plasmid). I transfected in a 24 well plate , changed the media after 6 hours post transfection. I...

08 September 2019 6,323 1 View

Max extension time for pcr with Phusion Hot start II DNA polymerase?

Hi all, I am planning to PCR a 7430 bp fragment from a big plasmid(11 kb) with Thermo Phusion hot start II DNA pol. Thermo recommend to use 15s-30s as extension time per 1 kb length. Overall GC%...

07 August 2019 3,134 3 View

How efficient and good is NEB Q5 Site Directed Mutagenesis kit?

Hello All, I just want to know your thoughts on NEB Q5 site directed Mutagenesis kit. Has anyone used this ? Is it good ? I have a plasmid of 6 kb on which I design my SDM . Your advice and...

05 June 2019 9,460 6 View

What is the maximum insert length limit for ligation?

Hi All, I am planning to clone one of my inserts into VC155 BiFC plasmid. My insert size is 8 kb where vector size is 5kb. Is it very hard to clone 8 kb into 5 kb? Also I would like to do...

04 May 2019 6,772 4 View

Why do we need at least 1 pmoles of DNA for Alkaline phosphatase mediated dephosphorylation?

I have been struggling with a cloning and its due to the fact that It is digested only with a single restriction enzyme(I have no other option). I was wondering why do we need 1 pmoles of DNA for...

03 April 2019 2,548 10 View

What is the alternative way to clean Ni-NTA column other than GuHCL?

Hi All, I need to wash and recharge Ni-NTA column. Unfortunately, I do not have GuHCL now. Can we use 8M Urea instead of GuHCL? Is it also necessary to wash recharged beads with Urea+ 0.2 M...

02 March 2019 4,673 3 View

His tag protein purification at Room Temp?

Dear All, Can we incubate Supernatant and Ni charged beads for binding at room temperature for overnight? Currently, we do not have a fixed temp room in our building. What about keeping Ni...

01 February 2019 6,831 6 View

Impact of N terminal His tag on signal sequence of secretory protein?

Hi All, I recently generated few constructs in pcDNA 3.1 HisC,encoding three different Fibronectin fragments.I did not realised that His tag was at the N terminal(I just overlooked). I...

10 November 2018 2,551 1 View

Can we store pierce glutathione agarose beads in 50 mM tris, 150 mM NaCl,pH 8.o ?

Can we store pierce glutathione agarose beads in 50 mM tris, 150 mM NaCl,pH 8.o after regeneration?

10 November 2018 7,178 3 View

Alkalaine phosphatase mediated dephosporylation procedure?

Dear All, This is going to be a very silly question to ask. I have digested my vector(~4000bp) with ApaI and I need to dephosphorylate to prevent self ligation. I looked at the NEB protocol for...

09 October 2018 4,023 3 View

To perform transfection with DharmaFECT Duo in AGS cells. Could you tell me what the ideal concentration is to avoid significant cytotoxicity?

I would like to perform transfection with the reagent DharmaFECT Duo (Horizon) on the AGS cell line. Could you please inform me of the optimal concentration to use without causing cytotoxicity in...

03 August 2024 3,851 1 View

Transfection in HEK293T cells?

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

Issues with Protein Expression in Transfected cells?

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

How to retain the a GFP tagged gene expression in stable cell line?

Hello , I established a stable cell line expressing GFP tagged to a centrosomal gene having G418 drug selection marker. I validated the stable line by IFA and Western blotting, results are fine....

29 July 2024 5,007 0 View

Does anyone have experience electroporating SKOV3 cells with a large plasmid using Neon transfection?

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

Why cannot i find my protein on cell surface after antibiotic selection of expressing plasmid?

I cannot confirm cell surface protein expression by flow cytometry even after transfection and antibiotic selection of cells. Does it take long for proteins to express on the cell surface? the...

28 July 2024 3,178 3 View

Can I use Polyjet after its expiration date?

I have a Polyjet that has passed its labeled expiration date for 1 year and I'm going to re-start transfection experiments. It is really needed to change it? Transfection efficiency may be lower?

23 July 2024 8,059 1 View

How to transfect circular mRNA to T cells?

Hello! We are using LNP to deliver circular mRNA into T cells. However, compared with linear mRNA, the transfection efficiency and protein expression duration are similar. Are LNP generation or...

21 July 2024 4,322 1 View

Transfection of A549 using lipofectamine 3000?

Im trying to transfect A549 cells using lipfectamine 3000. im using a 96 well plate with 1microgram plasmid DNA in each well . should i remove the culture media or change it or decrease it befor...

21 July 2024 7,610 2 View

Why there is a absence of colonies on the Abx plate for selection after liposome mediated DNA transfer ?

Hi All I am looking to get your opinions, I have perfomed liposome mediated DNA transfer into bacteria, the DNA is a plasmid with msfGFP and when I induce the plasmid I see the fluorescence, and...

16 July 2024 4,698 4 View