What is the best way to produce In-vitro RNA without the presence of +1G?

Most promoters used in the synthesis of RNA using in-vitro synthesis kit use +1G in their transcript. Is there a way in which this nucleotide can be escaped. Using RNase H may decrease the quality...

10 November 2018 5,022 0 View

Use of viral vectors with a GAP promoter, how do transcripts initiate?

10 November 2018 7,437 5 View

TSS data for wet lab researcher , easy bioinformatic analysis?

Dear coders and bioinfo enthusiasts, I wanted to elucidate the effect of nucleotide context on Cap proximal nucleotides in the 5' transcript leader. I wanted to look at existing TSS data from...

11 December 2017 4,607 0 View

Hi, Has anyone transfected cells with RNA and then isolated total RNA to reverse transcribe the RNA that was transfected?

I am trying to transfect RNA into cells. My transfection experiments seem successful as my luciferase readouts are to the tune of 100000 for 10 ul of cell lysate. When I obtain total RNA after...

31 December 2016 8,554 3 View

How do I do a pull down for measuring ability of certain RNAs to eIF4E/EIF4F complex?

I am planning to make small RNA molecules and cap them. I want to check the ability of these m7G RNA's to eIF4E/eIF4F complex, whichever may be easier. Has anyone done anything like this before...

06 July 2015 9,666 2 View

I am trying to make a cDNA library, but after RT when I circularize and PCR my library, I only see an empty library, can anyone help?

04 May 2015 5,122 6 View

What is the best way of filling in duplexes without amplification processes?

02 March 2015 8,285 7 View

What is the minimum intersection of template length required to anneal oligos? What is the best annealing buffer?

01 February 2015 1,574 7 View

What is the setting for obtaining a Polysome profile using a fractionator?

Hi, I am using a Brendel assembly of UA6 detector and Fractionator. As we dint have an engineer assemble it for us, we tried our hands at it. I wanted to know if there is a set program for...

01 February 2015 146 3 View

There is no commercial availability of cse3 enzyme. Does anyone have information on this?

Hi, There is a way to make the actual enzyme but the post purification step may involve getting an enzyme that is not active as the fplc in the department is not the best. Does someone have more...

01 February 2015 924 5 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

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

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

How to replicate the well conditions of a crystal hit with 20%(w/v) isopropanol?

I had a crystal hit in a well with 0.1M HEPEs pH 7.5, 10%(w/v) PEG 4000, and 20%(w/v) isopropanol. Why is isopropanol in w/v here if it's just solvent? How do I recreate this well condition?

25 February 2021 9,761 1 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