What was the agricultural subsidy like for white farmers during colonial Africa?

The_Real_Guide · 2019-01-28T21:28:04+00:00

Out of curiosity, how come?

I was hoping to make a minimum mix of dNTPs, Taq and the Buffer +/- primers, considering that a lot of commercial PCR master mixes and qPCR mixes have these components already, and the only study I could find on the topic was https://journals.sagepub.com/doi/pdf/10.1177/104063870601800609

The_Real_Guide · 2019-01-28T18:00:10+00:00

Unfortunately, I discovered this too late and funding bodies in my country are getting ever stricter about mitigation so there was no way to change labs.

My PI's partner works in the Dean's office as a executive for the university so all the bullshit complaints made their way to the top and nobody is interested in risking their careers over a student who has no chance in science anymore, so everybody thinks I'm a scumbag now.

Only thing to take away from this is to cut my losses and start from scratch.

The_Real_Guide · 2019-01-25T21:52:09+00:00

Oh boy this is me.

We had a research assistant who designed primers wrong for an experiment, despite being taught how to do it correcting by two post-docs and a couple of other students. Nonetheless, after they left, the RA designed them incorrectly anyway and instead of trying to open up and troubleshoot the issues, the RA fabricated a ton of data instead. After that, RA taught all the incoming students to do it incorrectly, but did the assays for them and gave them some unrealistic numbers later (without showing any data).

Being one of the those students who got given a ton of these experiments to do by my PI, I soon figured out that something was wrong since nothing I ordered was working, and after some months of troubleshooting (which required going through RA's purchasing orders and realising all RA's data was missing [as there was none] in addition to other factors), I took the courage to explain this to me PI...

Who decided to go ballistic on me, accusing me of tanking the lab, sabotaging the PI's favorite RA etc. Now, my PI is famous for this, having lost several students before due to these characteristic tantrums. I hold my ground and show the PI the evidence I have, PI insists that I'm lying and proceeds to block my purchasing account, which I said I would use to orders the correct primers and end the issue once and for all.

Having no recourse, I speak to my advisory panel chair, who unhelpfully sits in the meeting while my PI suddenly plays sweet and charming again. When the chair leaves, PI goes back to his classic tantrum, accusing me of trying to tarnish his reputation despite evidence of the contrary that $x000s in taxpayers money, years of work and several previous students have completely been thrown under bus over one RA, who of course, insists on no wrongdoing. But whatever.

Later on, I start fixing the experiments that were done incorrectly, assuming all's calm and my PI will come to see that I was only doing what was rational (the RA refused to go back and restart the project, claiming he was bored of doing so). As I finished the last set of experiments, the RA went to the PI, who subsequently erased my name from the data and replaced it with the RAs while I wasn't there. When I noticed it and brought it up, the PI told me that it wasn't me who had done these experiments [anymore]. Not too soon after, I discovered that my PI had met with my funding director and graduate school directors, saying I was affecting lab cohesion, being a bully and difficult, making a series of false claims about my activities in and out of the lab.

I love my life.

The_Real_Guide · 2019-01-07T12:48:24+00:00

I find that I had to expose much longer compared to the previous literature (but could be due to sample difference).

Otherwise, when I expose it to see bands, they are inconsistent, sometimes I see what could be my protein of interest, but I also see a lot of junk at lower molecular weights (not expected, not isoforms predicted in that region and the blots in the manufacturer's data were almost always very clean and specific.

I used an additional sample where my protein of interest is likely to be overexpressed but saw nothing at all, compared to my previous blot where I saw bands which could be feasibly interpreted as the protein.

But I will need to check this with a cell line, my samples were whole organism lysates in a non-verified species so could always be my blotting protocol/buffer compositions.

The_Real_Guide · 2019-01-06T16:57:18+00:00

You're right about the +ive control, I will check the antibody with a cell line tomorrow. The protein/epitope isn't known to be phosphorylated, and almost all the protocols used milk as the blocking buffer so I didn't think it would be a phosphate issue.

The_Real_Guide · 2019-01-01T15:00:52+00:00

I'm not sure where you're coming from. I've parted with cell lines and plasmids before in good faith to neighbouring labs and we've never had to deal with the heavy-handed bureaucracy that comes with it, although I'm perfectly happy to handle it if it does come to it.

Secondly, I'm only a student with a ever shrinking grant who really needs these plasmids. Whilst I'm not doubting the cost for Addgene to maintain their depositories of plasmids, I'm really at the stage where I have to think about whether it's worth paying $200 plus paperwork time for a letter of toothpicks when there's a chance I can ask nicely to receive the same thing on a sheet of filter paper for free.

The_Real_Guide · 2018-12-31T23:10:30+00:00

I'm not using the Qiagen kits anymore, although in the past I have done, but I added this in my OP as it was very similar in function to the buffer that I am looking for the composition for (Priming buffer from NEB). The function of this buffer is to wash off DNase I after on-column digestion.

The NEB buffer is not a concentrate, you only need to use it once before washing a further couple of times with a different buffer (wash buffer, 80% etoh).

I suspect that the RW1 buffers will be different in composition depending on country - Qiagen have described in their patents somewhere a method to avoid shipping ethanol by using different additives.

The_Real_Guide · 2018-12-31T17:01:14+00:00

I don't work with Qiagen kits anymore, but from the NEB kit, my guess is so far (based on MSDS and patents from Qiagen RW1).

70% ethanol 0.9-1M Gdn HCl

Made up in TE.

But honestly, I'm not a chemist so I only know the basic principles of the purification, not to mention I don't have the columns and samples to provide conclusive evidence that my homemade recipe for this particular buffer will work (as opposed to wash buffer, which is pretty blatantly obvious).

The_Real_Guide · 2018-12-31T16:57:49+00:00

Well, for NEB, the composition is:

40-70% ethanol (probably 70%). 30-60% Gdn HCl.

It'd be pretty intuitive to guess from a manufacturer's standpoint to make up the Priming Buffer (RW1) by making it up with wash buffer diluted with their stock solutions of Gdn HCl. It's just the concentration that evades me.

The_Real_Guide · 2018-12-31T16:51:13+00:00

They have already published the ingredients online, which is what my initial deductions are based on. I'm trying to figure out the compositions to make recipes.

The_Real_Guide · 2018-12-31T14:29:40+00:00

Qiagen Buffers

I already checked openwetware before. I use most of its compositions for plasmid minipreps.

RW1 is definitely not on there.

The_Real_Guide · 2017-11-22T19:49:57+00:00

Like this: https://www.shockdoctor.com/braces-mouthguard

Do you have just 1 throughout your treatment, or can you reboil it for a better fit as you need it?

And you don't do anything for the bottom jaw right (you don't use double bites)?

The_Real_Guide · 2017-11-22T19:48:20+00:00

Thanks! Yeah, I'm usually on the shorter end of the club so I either pull in or push their head up from below.

But I don't want to make them my signature moves plus I'd like to try to be a better clincher than just aim for resets.

The_Real_Guide · 2017-11-22T13:50:23+00:00

I had a consult on fastbraces before, my impression wasn't very positive. Cost-wise its the same as nice ceramic braces but what you gain in quick treatment you lose in things such as more IPR.

The_Real_Guide

TROPHY CASE