Can anyone suggest a recipe to make an Electroporation buffer to transfect CH12F3 cells ?

More Lakshani Perera's questions See All

How to clone the GOI into ROSA26 donor plasmid?

I am new to CRISPR Knock In. I want to clone a gene into ROSA 26 donor plasmid to Knock In a gene into the mouse genome. I have selected the addgene plasmid # 74285. But I cannot figure out where...

09 October 2019 9,551 1 View

Hello, I have a CRISPR related question. Can anyone help me?

I wanted to knock out MSH2 in mice cell line CH12F3 and after gRNA construction, transfection and single cell sorting, I got single colonies. I used two guide RNAs to give a deletion but when I...

04 May 2019 8,071 6 View

If I’m expressing a mammalian protein in bacteria, can I use an antibody for the protein that has species reactivity for human, for Immunobloting ?

for recombinant proteins do we have to use specific antibodies?

03 April 2019 9,791 3 View

How do I amplify a 7.4 KB fragment in mouse genomic DNA ?

I am struggling to amplify an 7.4 kB fragment which I need to confirm my CRISPR KO. I have tried Pfu and Phusion polymerase, used both HF and GC buffers, tried 50 - 70 C temp gradient, DMSO...

02 March 2019 1,401 4 View

How to design best primers to amplify a region in mice genomic DNA?

What is the database I should select under the Primer Pair specificity parameters in NCBI primer design tool?

02 March 2019 7,055 2 View

Has anyone tried using zeta probe membrane in DNA slot blotting?

I do slot blotting to my DNA samples that contain Carboxy rhodamine tags and then scan through typhoon (CY2) to get the signal intensity. Unfortunately membrane looks terrible and I cannot...

01 January 1970 2,877 0 View

Anyone familiar with DBCO -CY5?

I need to purify DBCO CY5 since it give lots of unwanted bands when run on PAGE. I am looking for a protocol for that.

01 January 1970 280 5 View

Can anyone suggest me a method to analyse altered replication dynamics other than DNA fiber analysis?

One of the compounds that we synthesized in our lab can result in replication fork collapse. We have tested for gmma H2AX foci that shows the ds strand breaks. But we need to test for the...

01 January 1970 6,431 0 View

Can anyone help me with this Urea-PAGE gel issue?

When I run my 17mer DNA oligos on PAGE gel I can see a smear above my DNA band. Does anyone know a possible reason or how to minimize this? I have attached a gel image here as well. Thank you

01 January 1970 928 4 View

Can anyone suggest me a suitable solvent system for Cl and azide separation where both are linked to n-hydroxyphthalimide with an ethyl group?

Cl and azide linked to n-hydroxyphthalimide

01 January 1970 1,847 2 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 anyone help me explain my problem?

Hi, Now I am doing a single-base editing by using HiFi Cas 9, guideRNA, single stranded DNA, HDR Enhancer V2 and my tranfection mehtod is lipofection (RNAiMax). Unfortunately, after gene editing,...

26 July 2024 2,136 0 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

Saleh Alkarim

Hello

Kindly look at in these links

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2975437/

http://www.jimmunol.org/content/jimmunol/early/2013/07/10/jimmunol.1300922.full.pdf

Good Luck

Christoph Metzner

There's a nice collection of protocols available at http://biorad-ads.com/transfection_protocols/

Best,

Lakshani Perera

Thank you all