Lauren Farwell

• Doctor of Philosophy
• PostDoc Position at University of Greenwich

Hello all,

I am quite new to performing qPCRs and am used to adding the primers in as a uM concentration.

A paper I am following states the following in regards to a qPCR reaction mix: "For the SYBR Green I based assay, iQ™ SYBR Green Supermix kit (Bio-Rad) was used in all reactions consisting of template DNA, 10 pmol of each primer (Clado-SYBRG-PF/R) and 12.5 μl of iQ™ SYBR Green Supermix in a volume of 25 μl."

I would like to calculate how much of the primer to add to my reaction in ul as a uM concentration. (e.g. 0.5ul of 0.1uM F primer). If you could show the workings so I can understand better, it would be much appreciated.

Many thanks,

Lauren F

Hi everyone,

We're running student groups where I'm based and our discussion for the next meeting is how to ask good questions after talks.

I know there are many researchers on here that attend conferences, seminars, etc, so I ask do you have any strategies, tips, or tricks on asking good questions?

I guess my big recommendation is to not be afraid of asking questions as it's likely other people are wondering the same thing, so what would be yours?

Many thanks,

Lauren

Hello everyone,

I have some questions in regards to using BLAST and sequencing data to give a species I.D. for fungal isolates.

We have created pure isolates of fungal colonies and then sequenced a gene region (shown in previous research to be a suitable DNA barcoding region for this genus) and ran this region (after sequencing both the forwards and reverse, creating a consensus sequence and trimming) through the BLAST database. While most isolates come back with mainly one species in the top search results, some have come back with various species in the top search results.

Therefore, how do researchers determine which species is the more appropriate I.D. using the BLAST database, or would you just say it is ambiguous based on the results?

For example, a general rule of thumb I was told in my undergraduate degree was you need an e-value below 10-4 and a percentage identity above 70%, however, if there are multiple species with similar values in the search results, how do you then decide which is most appropriate?

Is it purely based on the numbers, or would you look at the source of the result and preferably use isolate collections? Or, is it perhaps that newer species will have fewer entries in the database, so other species are likely to appear in the search results?

Thank you for your time,

Lauren

Hello everyone,

I will be performing a microbiome analysis and I wanted to know what the best method is to store the samples so the organisms stay alive?

From what I understand fungi are quite resilient so can be frozen at -20 degrees celsius, but bacteria are not.

Is there a standard method for freezing these samples to keep the cultures alive?

Kind regards,

Lauren

I have a response threshold that I have calculated using a sigmoidal curve fitted to my data set. What I want to understand is why this is a good method for finding a threshold over maybe setting a rule for when you detect a response? What makes a sigmoidal model good for generating threshold values? I have often seen it in the context of neural networks but am struggling to find it relating to biological thresholds.

Any insight would be appreciated

I am currently trying to understand the RNA extraction procedure. We used a plant RNA extraction kit (the E.Z.N.A plant RNA extraction kit from omega bio-tek) that involved cell lysis with a homogenizer column, addition of ethanol and then an RNA mini column that binds the RNA, and then we eluted the RNA using DEPC water.

What I want to know is how the RNA mini column only binds to RNA and not DNA? Is it something to do with the buffers having DNases so the DNA is degraded, or the column deferentially binds to RNA over DNA? Or does this depend on the kit used as to how only RNA is extracted?

Any insight would be extremely appreciated.