How can I remove a non specific band at a particular molecular weight in western blotting?

The protein of my interest is of 69 k Da, but i observe a nonspecific band at 55 Kda along with the specific band, even after increasing blocking i see this band, it also stains ladder at same...

08 September 2014 9,511 4 View

Can anyone suggest best transfection method (non viral) for SCC29B (buccal mucosa cell line)?

07 August 2014 5,380 3 View

Can anyone suggest me sequencing condition for a shRNA-plasmid forming strong hairpin loop?

sh-RNA for a gene is cloned in pLKO1.puro vector and it foams a strong hairpin loop, due to which sequencing reaction terminates abruptly. Please suggest me what can be done in this situation.

01 February 2014 5,347 2 View

Protocol for detection of Phospho EGFR by IHC

I am standardizing IHC for pEGFR detection using vector ABC kit, and tried different retrial conditions and antibody concentrations, but I'm unable to get staining. Can anyone suggest any specific...

06 July 2013 129 2 View

Which is best PCR primer for HPV (integrated or episomal) detection in oral tissues.

I use Nested PCR with MY09/11 followed by GP5+/6+ primers, but if anyone has a better set than these please let me know as prevalence of HPV in gingivobuccal cancer is very low, so I don't want to...

02 March 2013 9,480 5 View

Non specific band in PCR for HPV detection, what can be done about it?

I use PCR to detect presence of HPV in my tissue samples, In positive sample i get single specific band (150 bp) but those samples which are negative i get higher mol wt non specific multiple...

10 November 2012 5,914 29 View

Is it possible to determine the reason why one of my genes of interest is down-regulated, eg promoter methylation or something else?

In my study on human tumors, samples showed down regulation of one of my genes of interest. I am interested to know whether its because of promoter methylation or transcriptional inhibition. How...

09 October 2012 2,854 9 View

How to extract DNA and RNA from epithelium?

Normally I use QIAGEN kit for extractions from tumor tissues, but now I am planning to do it from premalignant lesions where I need only epithelial cells. I tried taking serial cryo sections and...

09 October 2012 8,145 21 View

What is the best protocol to treat culture with 5-aza-2'-deoxycytidine to block methylation?

I am planning to block methylation in my culture and check its effect on expression of particular gene, so how should i treat my culture with aza cytidine? i.e should I give continous dose or at...

09 October 2012 1,433 18 View

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Gel electrophoresis, RNA degradation, RNA extraction from fresh tissue

02 March 2021 5,433 5 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

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

Genome-wide gene identification protocol ?

Is there any book chapter/book, webpage or research article available to understand genome-wide gene identification?

28 February 2021 8,095 1 View

MiRNA qpcr problem?

Dear All, mirna primer showing some problem in the melting curve? any idea why? As attached is the melting curve. The forward sequence is obtained from miRBase and reverse primer is universal.

28 February 2021 5,008 4 View

Any specific pipeline or parameter to analyse total transcriptomics study of brain tissue samples?

I have two groups of brain samples, control and treated for example. It was total RNA nova seq sequencing. I tried all the available pipeline like: star+rsem+deseq2, Hista+stringtie+cuffdiff,...

27 February 2021 356 6 View

Why is the efficiency of viral mRNA electroporation in PBMC low?

Hello, I have been struggling with the electroporation of PBMC with HIV-I mRNA. Although the quality of the RNA generated is good, and the handling of the cells in meticulous, the frequency of...

27 February 2021 9,233 3 View

How to compare 2 samples recruited with different sampling methods?

If I want to compare characteristics of 2 groups of patients, one group is recruited from a random sample of the general public while another group is from a sample with a fixed gender (male to...

26 February 2021 3,526 3 View

Can MicroRNA cross the blood brain barrier?

In diseased conditions, many genes and gene products are visible in the bloodstream. So my question is whether we can find traces of microRNA which is involved in disease and disorder in blood serum?

24 February 2021 7,064 3 View

What is the best concentration of cDNA to be used for qPCR?

I have synthesised cDNA from 500ng/ul of RNA. The concentration of the cDNA Is about 5000ng/ul. Should I dilute my cDNA or just use it as it is for my qPCR? I usually use 2ul of cDNA for a 20ul...

24 February 2021 8,618 3 View