Gregory James Buchan

10 Questions 15 Answers 0 Followers

Questions related from Gregory James Buchan

How do I culture more CD11c+ BMDC from mice?

Hello, I am new to harvesting BMDC and my most recent experiment had 45% CD11c+ cells that were viable (also many dead cells, only 20-30% viable). I followed the method from Madaan A, et al....

07 July 2018 362 3 View

How many times can you strip a western blot?

Hello, How reliable are western blots after they have been stripped? Does the buffer strip all lanes equally? If you load 20 ug onto a gel and strip twice, how much would be left? I'm trying to...

02 February 2018 4,889 3 View

How much EDTA should be used for flow cytometry purposes?

Hello, I've been perusing the literature and a variety of protocols use EDTA concentrations from 0.03-5 mM EDTA in their buffers for isolating immune cells from the lung and/or BALF for flow...

01 January 2018 2,176 3 View

Why does the type of collagenase matter for lung digestion?

Hello! I am attempting to analyze cell populations in mouse lung homogenate using flow cytometry. I have seen a variety of protocols that use a range of collagenase and DNAse concentrations,...

10 October 2017 4,732 4 View

When propagating laboratory strains of Influenza, is ultracentrifugation required for purification?

Hello, I have been following: http://www.ncbi.nlm.nih.gov/pubmed/18770580 method for propagating PR8 in MDCK cells for our in vitro/in vivo studies. I have been collecting supernatants from the...

08 August 2016 8,114 0 View

How do you use cell lysates for studying enzyme kinetics?

Hello, I'm trying to determined the Vmax, Km, etc...for a membrane bound short chain dehydrogenase. I am planning on using cell lysates as a source of the enzyme. Do you know of any protocol to...

04 April 2016 1,989 5 View

What is the best way to determine the effect size?

How can you use preliminary data and/or previous research to determine your effect size so that you can estimate power and sample size required for future animal studies/clinical studies?

12 December 2015 3,342 3 View

What is a solid method to separate nuclear and cytoplasmic proteins from lung tissue?

I've been using commercial kits to separate cytoplasmic and nuclear proteins. I noticed that many advise against using their kits for frozen tissue because the nuclear membrane can break and leak...

07 July 2015 9,785 1 View

How does reaction time effect ligation?

Typical ligation protocols (for inserting a certain gene/deletion into a plasmid) recommend a ligation of 1-24 hrs. Has anyone seen significant differences in yield between 1 hr vs. 24 hr? Thanks

09 September 2014 5,699 3 View

Can anyone help with disappearing plasmids?

I want to know if anyone has ran into this problem before: I'm making a deletion construct and I have positive results in colony screening. When I miniprep the plasmid after overnight culture, the...

08 August 2014 4,125 8 View