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 9,506 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 8,042 6 View

No title

for recombinant proteins do we have to use specific antibodies?

03 April 2019 9,757 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 1,361 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 7,014 2 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 239 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 6,392 0 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 2,833 0 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 1,790 2 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 876 4 View

what is the simplest definition of ion exchange capacity

What this unit identifies for the ion exchange capacity ca. 0.92 meq. g-1. I am a newcomer in the membrane area. I just started. I highly appreciate your help

08 May 2021 0 0 View

what is the simplest definition of ion exchange capacity

What this unit identifies for the ion exchange capacity ca. 0.92 meq. g-1. I am a newcomer in the membrane area. I just started. I highly appreciate your help

08 May 2021 0 0 View

No title

Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

01 March 2021 5,311 1 View

Dapi fluorescence protocol for colon cell lines ?

28 February 2021 2,534 2 View

EMSA troubleshooting. Protein-DNA complex not migrating, cold probe lane with multiple bands. Can anyone help?

I'm running an EMSA to show the DNA binding activity of recombinant proteins versus the Wild type. Previous assays run by my research group using a different test system show the protein-DNA...

24 February 2021 8,325 1 View

Cloudy/high background western blot - can anyone help me?

Hi everyone! I try to detect VDR protein (~48 kDa) using Western Blot. After SDS-PAGE using 10% MP TGX Stain-Free Gel from BioRad I transferred the protein onto an metOH activated PVDF membrane...

23 February 2021 6,266 3 View

I see 2 random bands in my ERK/pERK blot? Has anyone else seen this?

I ran a western blot using 12% gel and probing for ERK/pERK (cell signaling antibodies). The green is total ERK, the Red is supposed to be phospho-ERK. I see 2 bands below the total ERK that...

22 February 2021 1,777 3 View

Protocol for membrane extraction E. coli?

Hello! I am interested in determining whether a poly-unsaturated fatty acid I have engineered E. coli to produce is being incorporated into the membrane of the cells or if it is simply a free...

21 February 2021 7,447 2 View

Any probe or assay for the detection of Cysteine in yeast invivo ?

Dear fellow researchers, I would like to detect the cysteine levels for yeast in vivo. I would really appreciate if anyone could suggest me any fluorescent probe or assay kit available. Currently...

11 February 2021 3,292 3 View

How can I label an antibody to determine how far it is travelling from the microdialysis probe?

I am trying to set up a new experiment where I am simultaneously utilizing reverse dialysis (antibody) through a microdialysis probe and a Miniscope for one-photon calcium imaging. Both pieces of...

09 February 2021 3,981 1 View