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Questions related from Tomáš Hluska
I'm growing Pichia pastoris in BMGY medium currently and I'm trying to follow, how it grows, so I measure OD600. But as I expected the yeast to grow (and hence increase OD600), I diluted it more...
29 July 2023 9,294 3 View
Hello everyone, I am once again transforming yeast Pichia pastoris after many years and have troubles again. I tried protocol by Kumar (2019) Simplified protocol for faster transformation of (a...
06 February 2023 7,837 2 View
Good morning, after some time, I'm doing again some cloning. This time I decided to try the DNA assembly to simplify things (how naive). However, at about that time I re-discovered this...
25 May 2022 938 0 View
Hello everyone, so I got this plasmid which I'm supposed to use, but I need to replace GFP with another fluorescent protein, so I decided to sequence it. Because I wasn't able to make much from...
12 May 2022 8,917 2 View
I'm doing some cloning after longer pause and I'm having some troubles with my DNA agarose gels. When doing colony PCR with QuickTaq HS DyeMix (...
23 March 2022 6,571 4 View
I think I've seen it previously, but the current example is from this publication https://www.mdpi.com/1420-3049/14/5/1825 where they used the following gradient on HPLC: min ... % MeOH 0 ... 2 20...
10 February 2021 5,565 3 View
I express my protein in pTYB12 vector as fusion with intein and then try to purify on chitin beads. Typically, I load overnight at low flowrates (something like 0.3 ml/min). The loading buffer is...
09 May 2019 8,639 5 View
Hello everybody, I'm currently growing E. coli in bioreactor and I have experience only with P. pastoris in bioreactor, so I'm not sure, how should it behave. With Pichia, one can try hard to...
19 March 2018 4,224 3 View
I have a phylogenetic tree containing approx. 2000 sequences. From those I have a selection of approx. 200 sequences. To visualize their distribution, I would like to "select" those 200 sequences...
06 July 2017 4,041 7 View
In my experience, we used Murashige&Skoog medium (either full or half) for Arabidopsis or tomato roots on Petri dishes, while Hoagland solution for other plants (as maize, maybe rice) in...
15 February 2017 9,371 7 View
I want to measure enzymatic reaction with nucleoside phosphates, stop the reaction with methanol and then measure the NTPs on C18. For the separation I need 1% or lower methanol concentration (I'm...
09 November 2015 8,953 6 View
I've run my 80% pure FMN on HPLC with fluorescent detector and I've seen at least six, maybe even nine peaks with the same exctitation/emission, as well as the same spectra on UV-VIS, so I...
23 September 2015 6,859 6 View
I use HPLC to measure FAD to FMN conversion by my enzyme. The conditions are as follows: A: 15 mM formic acid, pH 4.5 by NH3 B: MeOH column ZORBAX Eclipse Plus C18 RRHD 2.1x50mm 1.8 micron in the...
09 September 2015 7,555 5 View
Hi everybody, I'm talking about these http://tinyurl.com/Amicon-filters As I use them almost every day, I must re-use them. But since I don't want to lose my sample (obviously:), I always check...
05 August 2015 7,500 9 View
I have Pichia pastoris X-33 with pPICZalpha with my GOI. It's grown in bioreactor Biostat Cplus (Sartorius) in 2x YNB, 100 mM K-Pi,4% glycerol @28°C. I put the inoculum in on Thursday, since the...
28 March 2015 9,673 5 View
I'm about to transform Pichia pastoris with pPICZ alpha and pGAPZalpha with my GOI and because the Invitrogen manual doesn't specify the conditions, I'd like to ask you, what voltage do you...
26 January 2015 1,340 12 View
So the enzyme activity is calculated as either increase of product concentration [P] or decrease in substrate concentration [S]. In practise, it's determined often using e.g. spectrophotometer as...
22 November 2014 432 3 View
Hi everybody, I'm trying to grow rice because it has only one copy of my GOI as opposed to Arabidopsis. It plants do germinate (at least WT) and I got some seeds but it took more than 6 months and...
29 September 2014 9,395 9 View
My advisor told me he had some sheet with ad for pipette tips used to cut bands from gel. However he didn't have it anymore so he couldn't tell me the official name nor brand. Have you ever seen...
26 September 2014 8,694 7 View
I usually don't but since I'm about to use zeocin, I thought I'll make it "by the rules". However, because I do not remember what value it should be titrated to, I checked the internet and found...
03 June 2013 7,663 7 View
I noticed it already some time ago, that in accordance to suppliers MSDS some enzymes have actually higher activity in other-than-supplied buffer. Why do they not provide the best buffer? If you...
31 May 2013 9,183 3 View
Yesterday, I salted out my proteins (basically crude extract from maize) and dissolved in 1/10th volume buffer. I wanted to desalt it on HiTrap column so I filtered it through 0.22 um filter. When...
02 May 2013 5,545 14 View
I'm reading Guide to Protein Purification (From Methods in Enzymology series) and there is: "However, it must be pointed out that the concentration of the fluorophore cannot be calculated from...
19 April 2013 8,681 18 View
I'm studying protein crystallography now and I reached an inevitable point where I had to deal with the phase problem. Well, my problem is, that I'm not exactly sure what the phase is because no...
18 February 2013 1,725 5 View
I have experienced that when I run on HPLC for several days non-stop (either non-stop loading samples or alternation of loading samples and low-flow stand-by) that the back-pressure increases. I...
18 October 2012 8,565 60 View
I've found on Wikipedia, that the critical micelle concentration of sodium dodecylsulphate is 0.0082M, what is in rough agreement with value from this...
28 August 2012 7,008 4 View
I have been currently currently cloning one gene into pTYB12 vector and we had troubles with it all the time. Anyway, because we got no expression, I was currently trying to delete few first amino...
01 January 1970 5,962 4 View
After long time, I was doing again some PCR (if you wish to see how successfully, look here https://www.researchgate.net/post/What_is_wrong_with_my_DNA_agarose_gels-double_bands_and_weird_ladder...
01 January 1970 3,279 1 View
Hello, I want to do some cloning and for that I'll need to amplify the whole plasmid (~13 kbp), so I'm looking for DNA Polymerase which would have low mutation rate. We have Takara's PrimeSTAR...
01 January 1970 953 5 View
Hi, a friend of mine found the following question in preparation tests for medicine: How much water do you have to add to 1 ml of solution with pH 8 to reach pH 5? I assume they want simply...
01 January 1970 2,234 7 View
If I have a certain compound and I want to see, how is it metabolised in a plant, what are my options? Are there other options besides radiolabelled compounds? Are there commercial options to...
01 January 1970 5,823 1 View
