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Questions related from Tomáš Hluska
Hello, I'm trying to clone some short genes *up* to 350 bp into an expression vector the clasiccal way. For this, I do PCR with Q5 and get (mostly) nice band. I cut it from the gel and purify...
06 May 2025 594 10 View
Hello everyone, We are currently doing some deletions in one of our plasmids and after obtaining colonies, we are trying to do colony PCR, but it gives us just some smear (incl. positive control)....
13 November 2024 3,877 3 View
My PI wants to use expression plasmid for E. coli with two cloning sites. We have pColA-Duet-1 and pACYC-Duet, but in those are both of the sites inducible by IPTG (if I understood it correctly)....
14 October 2024 3,790 5 View
Hello, I sent 1 plasmid with 3 primers for Sanger sequencing last week, but all reactions were of poor quality. As the troubleshooting suggests to almost every problem to send PCR fragment...
02 October 2024 723 8 View
I've sent my plasmids for whole plasmid sequencing using the Oxford nanopore sequencing method. All of them were assembled as monomers, but in the reads (see attached pictures), there are even...
20 June 2024 1,698 5 View
Last week, I transformed home-prepared E. coli TOP10 cells. When checking the colonies, I noticed that some of them have yellow color. They did not grow on the randomly, but dependently on the...
17 June 2024 3,650 3 View
A friend did PCR and then loaded it on agarose gel. All samples were prepared in the same way; the PCR was identical, with the same template, only primers differed, the same amount loaded on the...
05 June 2024 3,129 3 View
I did PCR of 6 amplicons and when I did agarose gel electrophoresis, they all appear consistently bigger than they should be (e.g. the amplicon should be 4000 bp, but is around band of 5000 bp...
10 April 2024 4,568 5 View
I just found the Platinum SuperFi II DNA Polymerase, which should simplify PCR protocol as it allows uniform annealing temperature of 60°C, but it should also have very high fidelity; >300×...
18 March 2024 7,754 4 View
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 9,410 3 View
I have an Arabidopsis protein, which should be apoplastic (rice homologue is localized to apoplast and this protein contains predicted signal peptide). I expressed the protein without the signal...
21 June 2023 2,443 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 7,952 2 View
I'm going to transform Pichia using the LiCl transformation protocol from Invitrogen. In that, they use PEG-3350. We have PEG-4000. Would it be OK to use it or do I need to order the PEG-3350?
05 October 2022 2,289 3 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 1,011 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 9,011 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 6,673 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 5,783 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 8,731 5 View
Hi everybody, I'm currently expressing proteins in and extracting from bacteria. The procedure is that I pellet the bacteria from bioreactor, resuspend in buffer (50 mM Tris/HCl, pH 8.0; 1M NaCl;...
17 August 2018 8,051 9 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 4,298 3 View
It seems impossible to find on the internet, so far no luck.
29 August 2017 5,877 6 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 4,144 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 9,515 7 View
Older publications claim, that starch synthase can utilize UDP-Glc as substrate (e.g. Baxter and Duffus 1973 http://link.springer.com/article/10.1007%2FBF00387476 you can see the first two pages...
26 April 2016 9,659 3 View
I'm currently using triethylamine for separation of nucleotides on RP HPLC columns and they have very, very short lifetime. I was recommended to use either double end-capped (as Zorbax XDB) or...
22 March 2016 8,826 3 View
The FPLC columns usually have specified some recommended flow rate, but for (U)HPLC it's common to have only maximum pressure, if at all. So how do you choose the flow rate? Do you always go for...
26 November 2015 9,381 11 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 9,030 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 6,940 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 7,630 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 7,575 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 9,753 5 View
I have some plants with altered enzyme expression and I'm interested in following compounds: flavins (mainly FAD and FMN, not sure whether it's possible to discriminate OR to include those bound...
17 February 2015 7,322 4 View
Why is the DNA used for many purposes like carrier DNA for yeast transformation specifically from fish AND their sperm? Is it available in such big quantities and easily obtained? Why not DNA from...
03 February 2015 535 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 1,415 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 545 3 View
Hi everybody. I want to try to grow Pichia at various pH in flask to see what will be the best for their growth AND my protein. I want to try range about 3.5-4.5 and 6.5-7.5, because my protein...
05 November 2014 5,889 1 View
I went through my first harvest of the rice and although I got some seeds from the WT plants, most of them were initiated (i.e. the pollen made it in etc.), but then they were aborted and the...
03 November 2014 8,114 8 View
So I have an enzyme with pI 5.5. I'll try to produce it in Pichia in medium with pH 4.0 or 4.5 as it grows better. However, as I perform purification in basic pH (7-8), I want to change the pH of...
26 October 2014 3,959 11 View
I'm trying to run fermentation of Pichia pastoris using the AOX1 promoter but so far I didn't get any activity (it's positive clone from shake growth). Can somebody tell me how much...
20 October 2014 3,969 14 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 9,462 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 8,767 7 View
I have problems growing Arabidopsis on MS plates. The plants are small, reddish and only small fractions of plants germinate. I have tried it by myself (previously a student did it) and it's...
11 May 2014 1,309 33 View
Since Triton is a non-ionic detergent, it should not bind to ionexes such as ResourceQ, right? So in principle it should be possible to use ResourceQ chromatography to get rid of excess Triton, right?
06 April 2014 9,782 5 View
I'm currently fighting with the Strep-tag/Strep-tactin chromatography. I observed, that the HABA is not eluted completely even by 100 mM Tris base (pH ~10.5). After the loading of proteins, I...
14 February 2014 4,493 13 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 7,742 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 9,252 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 5,611 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 8,748 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 1,797 5 View
In the first table in attached link are listed pheophytins corresponding to chlorophylls. However, Wikipedia speaks about pheophytin as about single compound. So I want to check, whether is it...
12 November 2012 111 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 8,667 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 7,085 4 View
I'd like to make some analysis, but I don't know anybody with such equipment. Do you have access to such or know about anyone else? If so, please, let me know.
13 July 2012 1,839 3 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 6,054 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 3,361 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 1,054 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 2,314 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 5,893 1 View
Hello everyone, I'm working on a protein, homologue of which is associated with cell wall (I don't know about my protein yet, but it should be in the cell wall as well). I expressed this protein...
01 January 1970 562 1 View
I have been reading about various fluorescent proteins and most of the work seems to come from Japan. Are there any groups who would study, discover and/or improve fluorescent proteins based in...
01 January 1970 4,521 1 View
When I feed DHZ (a plant hormone cytokinin) to pea plants, I observe a novel peak on HPLC. I don't know what it is and I'm planning on identifying the compound, but in the meantime I'd like to...
01 January 1970 1,656 0 View
I want to confirm protein localisation in Arabidopsis using fluorescent protein-tag. What are proper markers for plasma membrane, apoplast and cell wall? Would anybody share such lines or plasmids...
01 January 1970 8,607 2 View
When I feed a particular substance to pea plant, I see conversion to unknown compound. I don't know, what is the product, I don't know (obviously), what is the reaction and I do not know required...
01 January 1970 8,240 2 View
I started working with RiPPs (Ribosomally-synthesized and post-translationally modified peptides) in cyanobacteria recently and I'm wondering. All the papers I have found so far deal with their...
01 January 1970 2,004 1 View
There has been (relatively) recently published a paper ( https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219008 ), where they claim they found QTL for ratio between two...
01 January 1970 4,500 0 View
I'm trying to clone some large pieces of cyanobactrial gDNA (sometimes even more of them) into a plasmid via fusion PCR. Then, I transform it into E. coli TOP10, as my PI says it works better...
01 January 1970 5,610 13 View
Long time ago, I read that the culture for plasmid isolation should be fresh for best results. I took from that that it should be actively growing and was trying to get such cultures since then....
01 January 1970 8,966 5 View
I'm doing recetly plenty of cloning and to find E. coli colonies with my desired construct, I do colony PCR. However, as I am preparing brand new constructs, using new primers for the colony PCR,...
01 January 1970 498 10 View
