I did like to know what to do to get a PCR product that will come out with a single clear band, no noise and mixed peak when the product is sequenced?

I am trying to check the polymorphisms of a particular gene from human DNA samples. A set of primer in a published publication allowed the amplification of the DNA hence permitting the analysis of...

02 March 2019 8,268 5 View

What could be the cause of noise or mix in PCR sequence result?

I subjected some genomic DNAs to PCR reaction and later sent then for sequencing to analysis for polymorphisms in my gene of interest? Some of my sequence results came out fine while some came out...

01 February 2019 9,564 7 View

Deglycosylation test: I can't understand why the bands from my deglycosylated proteins are not neat?

I carried out a deglycosylation test to see if there will be difference in the electrophoretic mobility of C57BL/6 infected-ME7 and SShpPrP Tg ( transgenic mice expressing sheep prion protein)...

01 February 2019 5,754 2 View

My transfected cells do grow at sporadic rate a week after subjecting them to antibiotic selection treatment , what could likely cause this?

I am trying to establish a stable cell line expressing a gene of my choice using a prion protein knock out cell line, Lipofectamine and Zeocin as selection antibiotics. Not ruling out the fact...

11 December 2016 8,981 7 View

What could be the likely causes of false positive or false negative results during PCR reaction?

I did tail biopsy to genotype a particular gene. The result came out positive for my desired gene but two days after using same lysed genomic DNA, it came out negative. In my confused state, l...

11 December 2016 5,573 7 View

Please l need some one to help me with protocol to establish cell line using Lipofectamine LTX plus reagent and G418 as antibiotics for selection?

07 August 2016 6,533 2 View

Please help with expert views based from experience on establishing cell lines expressing prion protein gene from any of the cervids- elk, deer ?

I need help on the most appropriate transfection approach and conditions to optimize cell transfection when establishing stable cell lines expressing any of the cervids' prion protein genes and...

06 July 2016 2,462 0 View

For BCA or brain homogenate preparation, what volume of Lysis buffer should be added to weighed cell pellet or tissue when lysing cell or tissue?

06 July 2016 4,526 0 View

I did a transient transfection on Zpl neuronal cell line but the transfection wasn't successful. Please l need help on what could have caused this?

I am trying to establish a cell line using Zpl cell line- PrP knock out and LTX/PLUS lipofectamine reagent as transfection reagent.

06 July 2016 247 4 View

Can Micro RNA be extracted from tissues?

I need protocols or procedures for extracting Mi RNA from tissues in experimental animals.

04 May 2016 1,103 4 View

Why does blocking with my immunogenic peptide cause nuclear fluorescence in ICC?

Hi all, I am validating a polyclonal antibody raised against human ANT4, through WB and ICC. In most of my controls, the antibody shows very little off-target binding, but when I pre-incubate the...

03 March 2021 5,999 3 View

Have someone successfully used the ER alpha (Santa Cruz sc-8002) antibody in immunostaining ?

Santa Cruz now recommends this new antibody (F-10) but I can't find a publication using this reference. I'm working on human et non human primate hypothalamus and so far, it doesn't work. Is...

02 March 2021 10,003 2 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Does anyone know an alternative product for cardiac Troponin T antibody for Immunofluorescence (Thermo # MA512960)?

We have been using Thermo Fisher anti-cardiac Troponin T antibody for immunofluorescence staining in FFPE mouse heart sections but it was recently discontinued. The literature shows it to be the...

01 March 2021 4,937 1 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

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

Precipitate after adding laemmli buffer to protein solution containing Potassium Acetate?

Hi, I am running a size exclusion chromatography experiment with a buffer containing Potassium Acetate as a salt. I analyse these fractions through SDS-PAGE. After boiling my SEC fractions in...

01 March 2021 2,622 3 View

What is the most suitable server for predicting protein structures in bacteria?

I have used the i-Tasser several times, however it has been unavailable for several days. I tried the swiss-model, but the output was not very pleasant due to the model used. Is there any other...

28 February 2021 4,521 3 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

Are protein samples in SDS compatible with ELISA?

I have used an AllPrep DNA/RNA/Protein Mini Kit QIAGEN kit to extract RNA and protein from my samples. At the end of the protein purification, I resuspend my protein in 5% SDS. Will these samples...

28 February 2021 7,370 3 View