Multiple sgRNA cloning into pX333 plasmid using BsaI and BbsI restriction enzyme site?

Currently, I am using a plasmid containing two gRNA cloning site. The restriction enzymes used for cloning gRNAs into this plasmid, pX333, are BsaI and BbsI. I digested the pX333 plasmid using...

09 October 2019 2,743 6 View

How do I design primers specific to transfected plasmid and not genomic DNA?

I recently introduced a Lenti-Puro Guide vector containing a specific sgRNA sequence into a cell line. I would like to sequence the ddCas9-sgRNA cell lines to confirm that each sgRNA is accurate.

01 February 2019 5,376 3 View

Has anyone uses a 4in1 cholesterol tester on research mice?

Hi all, Has anyone used a 4in1 cholesterol tester on research mice? We have used the One Touch handheld glucose monitoring system to measure glucose on high fed diet fed mice. However, has anyone...

09 October 2016 3,866 2 View

From a budding scientific illustrator and PhD student - Are you in need of a scientific illustrator?

Are you in need of a scientific illustrator for creating illustrations (cellular and molecular mechanisms, structures, etc) to compliment a primary paper or review article? If so, please let me...

07 August 2015 5,419 0 View

Should I serum starve primary hepatocytes if I wish to probe for pAMPK using westen blotting?

I am using stimulated primary hepatocytes to detect alterations in AMPK activity. However, a paper published that serum starvation can increase AMPK activity in different cell types. Although,...

07 August 2015 7,940 3 View

Does anyone know of easily transfected mouse cell lines?

I would like to assess the editing efficiency of designed guide RNAs by transfection prior to establishing a stable KO cell line (via lentiviral infection). HEK293T cells are very efficient when...

01 January 1970 6,434 2 View

Interested to stain mice tissue with WGA 488 conjugate, anybody has a protocol to recommend?

Interested to stain mice brain tissue with WGA 488 conjugate, appreciate if anyone willing to offer me some guidance or suggestions! Thank you!

28 February 2021 3,951 2 View

B-mercaptoethanol (BME) protocol?

Hi everybody, I have been running a multiplex IHC which allow me to stain the same tissue with different markers (Primary Ab + Secondary conjugated Ab) (AF487, AF555, AF647). Since I would like to...

27 February 2021 5,028 2 View

Why do my MCFs not form monolayer?

Hello, I am looking for some expertise on culturing MCF7s. I purchased them from ATCC and I have been following all media and culture conditions. Media is EMEM with 10% FBS and 10ug/mL of...

27 February 2021 3,904 2 View

Is there anyone who experienced with cytotoxic effect of Hoescht 33342 dye?

Is Hoechst toxic to cells? In general, it is known as non-toxic. I am working with dsRED MDA-MB-231 cell line. I applied it for my PDMS-based microfluidic platform, for this reason, I incubated...

26 February 2021 9,119 3 View

Why my agarose gel staining sometimes looks not homogenic but like gradient (see the figure)?

I am worried about this overexposure of the upper part of the gel in the picture. Is it possible that this is the result of too much concentration of ethidium bromide? Why is there such a big...

25 February 2021 8,140 3 View

Markers of mitochondrial activity in FFPE tissues?

Hello everybody, Does someone have any experience with assessing/extimating mitochondrial activity on FFPE tissues? Precisely, no TOM20, ATP5B, etc: we are trying to get an approximative idea of...

24 February 2021 4,618 4 View

Can a Golgi-stained dendritic spines picture processed by Image J be directly used for publication?

24 February 2021 3,348 2 View

Is there an appropriate CD8 antibody for IHC-P for staining human breast cancer tissues?

Hello, We are planning on staining Formalin-fixed paraffin breast cancer tissue sections (FFPE) with CD8 Ab via immunofluorescence (Tissue IF). Based on our existing protocol and imaging...

24 February 2021 9,578 4 View

Reagents for histology slides?

Dear all, I am planning to study histology and want to do that at home. I have only stained a blood smear with Giemsa but now want to try a wider range of animal tissues. What reagents will I...

24 February 2021 5,430 3 View

Are there lab markers/pens resistant to Roti-Histol?

Hi, I'm looking for a pen/marker to write on the smooth, transparent (non-frosted) part of microscopy slides which does not get washed away by alcohols and most importantly Roti-Histol. I want to...

24 February 2021 9,902 5 View