RNA extraction question

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Immunostaining in SSC 2X buffer?

Hi, I would like to know if some people use SSC 2X buffer for immunostaining, i should do an experiment where i cant use PBS in drosophila embryos. If you have advice or knowledge about Staining...

03 April 2019 2,683 9 View

Drosophila embryo permeabilization to deliver drugs and image at the same time?

Hi all, I want to image drosophila embryo with drug treatment, of course the major problem is the viteline membrane, and the survival of embryos to permeabilization. I wonder to know if someone...

02 March 2019 251 2 View

Immuno smfish drosophila?

Hi, I am coming here because i tryied several protocol To do an immuno single molecule fish in drosophila embryos and it works with some probe by doing fish follow by immunostaining but i recently...

31 December 2018 5,257 4 View

Visible immunostaining using DAB ?

I wonder to know if immunostaining using DAB is sensitive to amount of tissues as il use drosophila embryo i cannot know exact amount of tissues and for some mutant i have very few embryo. Is this...

01 February 2016 188 3 View

How to normalize an RNA immunoprecipitation?

I have a problem with the logic of an RNA IP normalization. I am comparing two RNA binding proteins tagged with GFP, one is an isoform of the other where I mutated one binding site. My IP is done...

04 May 2014 1,942 3 View

When you extract RNA with Trizol you should break the RNA if you vortex too much ( >1min.)?

If it is the case is it a problem for qrtPCR after that as I do reverse transcription with a random hexamer. Thanks for your answer.

31 December 2013 4,814 4 View

Does anybody have a protocol for reverse transcription?

I have tried various different protocols for reverse transcription with random primers and I don't see any obvious difference in the final qPCR reaction. Any suggestions? I would also like to...

31 December 2013 5,078 1 View

How do I increase the yield of my PCR product?

Hello, We would like to increase the yield of our PCR product. We are running a series of PCR reactions that is targeting ~1.1kb sequence. We begin each reaction with ~400pg of template DNA...

02 March 2021 4,029 3 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

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 View

Does anyone know HRM channel in a 2 plex QIAGEN Rotor- Gene Q qPCR can be used as a separate channel?

I am going to have 3 different probes in my qPCR work that I am going to do. But I realized that the machine we have in the lab is a Rotor-Gene Q 2plex HRM Platform, saying it has green, yellow,...

01 March 2021 8,544 1 View

How to address this issue in the analysis of Real-Time PCR results?

To dear Researchers, I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8...

01 March 2021 8,683 4 View

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Does anyone have the experience of using Taq Man probes in the QIAGEN Rotar- Gene qPCR machine?

01 March 2021 5,311 1 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

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

Binal Shah

If all you are changing is the g-force, it should not be a problem. OTher than you may have to spin for slightly smaller time as you have a higher g force. You may also precipitate some salts, which you can wash off with alcohol.

Jeremy Dufourt

Thanks a lot for these answer

After discution with invitrogen technical support they say that i can centrifuge at more than 16000g and for a longer time this could improve rna yield but the rna pellet is harder to dissolve.

Jack M Gallup

Heating the pellet (after adding the desired amount of nuclease-free water to it) to 58-60C for 2 or 3 minutes will also aid in resolubilization if you suspect you've over-dried.