Irina Afonina

9 Questions 14 Answers 0 Followers

Questions related from Irina Afonina

How can I compare mRNA and protein stability at 46 C?

Expression levels differ for two proteins at high temperature in E. faecalis. Might be just due to instability of longer mRNA or larger assembled protein for one of the proteins. Are there...

09 September 2016 1,438 0 View

How do I distinguish between released bacterial membrane vesicles and those formed from the cell debris?

An interesting point was raised during MV discussion. We do know that bacteria produce membrane vesicles which can be isolated from the supernatant by ultracentrifugation and gradient...

04 April 2015 9,245 10 View

How can I prepare Western blot samples from vesicles?

I'm going to isolate vesicles from bacterial supernatant and run western to see if my protein was incorporated into them. After ultracentrifugation I obtained pellet which supposedly contain...

12 December 2014 7,851 2 View

How can I analyse qPCR data with different efficiencies?

I compare control with treated sample and use single HKG First I was using ddCt method, but then decided to switch to more precice Pfaffl method to include my efficiencies which are not the same....

11 November 2014 7,274 9 View

Are bacterial cells fixed in 4 % PFA still alive?

I'm going to view localization of certain fluorescent protein in Enterococuccus. As they tend to form chains, and for analysis I need to use single cells images, I wonder if I can fix in first...

02 February 2014 3,891 2 View

Automatic threshold for qPCR analysis?

Do we need to adjust threshold to certain value or select specific one and use it throughout the experiments? Such as if we compare two samples treated/untreated we set same threshold (will it be...

01 January 2014 1,511 5 View

Can I use both gyrase A subunit and gyrase B subunit as housekeeping genes for qPCR?

For downstream analysis in gene expression, it is recommended to have at least 2-3 housekeeping genes. May I use gyrA and gyrB as well as a third gene, or as they encode for same enzyme but...

12 December 2013 6,767 2 View

Will primer optimization help with qPCR amplification in negative control after Ct30?

I'm optimizing assay: 7 genes, efficiency 93-103%. For 3 of these genes I have consistent amplification in Negative (NFW) control. I'm using SYBR, so it might be due to dimers formation? When...

12 December 2013 5,059 5 View

2-Steps RT-PCR, cDNA concentration and quality

If I do cDNA synthesis with SuperscriptIII using 2 mg of total RNA in 20 ul reaction, do I need to quantify cDNA by nanodrop and optimize concentration for RT-PCR, or can I just use 0.5 ul of cDNA...

11 November 2013 304 9 View