Can we precipitate RNA alone but leave DNA in aqueous solution?

Assuming I would like to use some chemical mediated process of precipitating RNA and polysacharides (from tissue lysate) but leave genomic DNA intact in the aqueous layer. What are the best...

06 July 2018 2,757 3 View

Can we store taq polymerase, respective buffer, primers, probes together as single constitution?

Many commercial diagnostic kits send single reagent (Premix) to which the user must just add respective DNA and perform Real Time PCR. What are the key parameters to be considered while preparing...

05 June 2018 5,717 7 View

What could be the reason for no signal at all in Taqman FAM-BHQ1 probe and HEX-BHQ1 probe?

I have two targets A and B both are known sequences cloned into plasmids, The primers Tm for A is 54 and respective probe Tm is 63 The primers Tm for B is 59 and respective probe Tm is 69. PCR...

05 June 2018 6,152 7 View

What if the distance between Sense primer and Sense probe is more than 90 bp?

We have an issue with the Taqman Probe (FAM-BHQ1) based real time detection of target. What we have been observing is at higher copy numbers >2Crore we are getting very good amplification plots...

03 April 2018 7,143 6 View

Which is the best inhouse protocol for midi and maxi prep plasmid DNA isolation?

Looking for midi and maxi plasmid isolation protocols from E coli (high copy number plasmid) cultures above 100ml (LB) Protocols tried so far: Sambrook's midi and maxi prep Alkaline lysis...

03 April 2018 183 4 View

What can improve copy number detection sensitivity of real time pcr FAM-BHQ1 probe based?

I am working with development of viral diagnostic kits using Real Time PCR (Probe based) detection. As per standard procedures we do use plasmid carrying viral gene as control and to determine the...

10 November 2017 2,666 22 View

How to calculate copy number for a plasmid and make necessary dilutions. Thermoscientific tool?

How can one calculate the copy number with following details: plasmid size 4000 bp concentration 1600 ng/ul Now I did try using thermoscientific online tool which does tell u total copy numbers...

07 August 2017 5,549 1 View

What are the strategies to design a 1 Kb in house DNA ladder?

I am looking for strategies which most of the commercial companies use for production of 1 Kb DNA ladder. I did come across good journal for a 100 bp ladder where they have designed primers using...

07 August 2017 7,624 4 View

How to asses the efficiency of bisulfite treatment of genomic DNA?

To perform a methylation specific PCR one has to treat the DNA with bisulfite. But are there any standard measurable ways to determine to how much extent the conversion of unmethylated Cytosine to...

11 December 2016 3,901 2 View

Determine hypo and hypermethylation state using Real Time PCR?

11 December 2016 7,757 1 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

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

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

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

Recommendations on homogenizers for RNA isolation?

I will be performing RNA isolation with frozen lung biopsy samples and am in the market for a tissue homogenizer. I am wanting a homogenizer that will fit in a 1.5ml tube. I was looking at the...

24 February 2021 3,623 3 View

RNA-seq DEGs downstream analysis and reporting in literature?

I used EdgeR to get the list of DEGs (differentially expressed genes) from my liver RNA-seq experiment. I have done the gene overrepresentation and GSEA based pathway analysis. I am currently...

23 February 2021 8,969 1 View

Is it efficient to do liquid biopsy to investigate the gene expressions of the tumor?

To do liquid biopsy and look for specific gene expressions there are so many published papers. Thera are different protocols and procedures used to isolate circulating cell free RNA or mRNA from...

23 February 2021 7,326 5 View

RNA degraded after good nanodrop readings?

23 February 2021 8,850 2 View

Is there a way to extract total RNA and protein from cell culture at the same time, without denaturing protein?

Hi all, Is there a way to extract RNA and protein from the same cell culture sample? Because we need to use the protein for gelatin zymography at later time, we are looking for a method that can...

22 February 2021 5,341 5 View