Amila Zuko

11 Questions 24 Answers 0 Followers

Questions related from Amila Zuko

How to avoid uneven (intensity) bands during Western Blotting?

I've uneven bands (size, intensity, and staining) when doing Western Blotting and I'm wondering what the problem could be. The protocol: -10% separating gel (0.375M Tris pH8.8; 0.1% SDS; 10%...

15 July 2019 6,874 8 View

How to choose dendritic area for spine measurements in primary neuronal cultures?

Several papers I've read don't mention what area is selected, making it look random. Other papers mention a particular dendritic branch or distance from soma. Is there a consensus on this? What...

18 April 2017 2,370 4 View

Screening knock-in mice (by CRISPR/Cas9)?

I've performed microinjections to create a tag-insertion inside my gene of interest by CRISPR/Cas9. A quick PCR shows that a bunch of the pups are positive. However, all are heterozygotes. I'm...

23 February 2017 6,506 2 View

Has anyone tested crRNA and tracrRNA in vitro (cell culture)?

I want to see whether the crRNA I've designed works in vitro (cell culture). I've successfully performed in vitro digestion of 5 different crRNA's + tracrRNA and I've selected the most efficient...

22 December 2016 1,482 20 View

How to reduce the amount of coeluates after IMAC?

I'm performing IMAC to purify my his-tagged protein (enzyme) from e. coli lysate. It seems I can get a good amount of my protein of interest but the problem is that I also get alot of coeluates,...

13 October 2016 9,960 7 View

Failed Histagged-protein purification from E.coli, sonication problem?

I have tried 6xhistagged-protein production and purification from E.coli but nanodrop couldnt detect any protein in eluates from nickel-resin beads. I suspect my problem is sonication: I spin...

29 July 2016 1,811 16 View

What is the difference between the functions of N2 and B27 supplements during ES cell differentiation into neurons?

Several papers base their method of ES cell differentiation into neurons on Bibel et al., 2007 (Nat Prot). When the cells are initially plated for neuronal differentiation the medium contains N2...

23 June 2016 6,792 4 View

How to dissolve PEI (polyethylenimine)?

This concerns linear PEI 25kDa. So far I am aware of two methods: 1: stir the PEI in MQ (1mg/ml) and dissolve by dropping and holding the pH at 2. After its transparant increase pH to 7 -->...

21 April 2016 3,098 22 View

What could be the reason for these strange chromatograms (see attachment) after DNA sequencing?

Lately I'm getting failed DNA sequences and strange sequence peaks in my chromatograms when trying to sequence my plasmid (see attachments). With a previous mini prep sample, the following...

09 February 2016 6,594 4 View

Which neuronal precursor cell is best to transplant into mouse cortex to study neuronal differentiation?

I would like to study the development of single cells (neurons) that were treated in vitro before transplantation into the mouse cortex. At the moment I have mouse embryonic stem cells available...

19 November 2015 9,762 4 View

Could someone help me with some Neuronal Tissue Co-IP issues?

I have been doing co-immunoprecipitations for a while now using my own buffers (CHAPS lysis buffer + protease & phosphatase inhibitors) and Dynabeads protein G beads. Useful information: -The...

22 January 2014 5,393 3 View