Julia Mawer

8 Questions 10 Answers 0 Followers

Questions related from Julia Mawer

How do you recover your yeast cells when you transform them with a plasmid that has the KanMX4 resistance?

When I transform linear PCR products that contain the KanMX4 cassette, I usually plate on YDP, incubate overnight, and then replica plate on G418. This works a charm. However, this time, I am...

18 March 2020 9,775 4 View

Has anyone had problems with Dynabeads and background binding recently?

Hello, I have a very odd problem. A few years back (2016) I was using MyOne Streptavidin C1 dynabeads and coupling them to a biotinylated peptide and doing pulldowns using whole cell lysates. My...

10 October 2019 8,555 5 View

Can you see the pellet when you wash a TCA protein pellet in Acetone?

I am following standard protein precipitation. Mix 1:1 sample and 50% TCA for a final TCA of 25%. Ice 10 min then centrifuge 14K RPM 4 degrees 5 min and I see a lovely translucent pellet (30ug...

26 March 2019 4,082 3 View

What is the best way to add a nuclear localisation sequence to the N-terminus of a protein?

I do not have a plasmid containing the NLS. I was planning to add the NLS to the sequence of a primer. My question is: Do I just place the sequence straight after the Start codon without a linker...

30 August 2016 875 4 View

Which budding yeast nuclear membrane protein is best to tag for fluorescence microscopy?

I already have a GFP-tagged nuceolar-binding protein and I wish to visualise this with respect to the nuclear membrane. What is the most common nuclear membrane protein that people tag?

06 July 2015 4,831 3 View

Has anyone isolated mononucleosomes (following MNase digestion of chromatin)....ideally from yeast?

I have a protocol for digesting yeast chromatin with MNase and when I run the DNA on a 1.5% agarose gel, the majority of my species are indeed in the mononucleosome form. However, I wish to do an...

12 May 2015 6,113 3 View

What is the best way to strip a Western Blot?

Does stripping with only glycine, SDS and tween tend to leave a background signal?

10 December 2014 2,967 4 View

Should you add DTT to the lysis buffer for a CoIP?

I am aware that not adding a reducing agent to Laemmli buffer before elution can reduce the amount of antibody contamination in your samples. However, I would have thought that DTT should not be...

28 November 2014 2,755 5 View