26 Questions 15 Answers 0 Followers
Questions related from Maria Victorova
Dear everyone, I work to improve the function of IRESes to make translation of mRNA and circRNA more efficient. The IRESes we are using are not very strong. I already looked at IRESite. If you...
16 February 2023 1,243 2 View
Dear colleagues, Could you please suggest the best way to thaw and freeze purified mRNA in water. I have batches of unencapsulated mRNA in nuclease-free water supernatant from nanoparticles and...
10 August 2022 3,229 3 View
Dear colleagues, I have applied 1 ug/mL of doxycyline to human iPSCs but most cells died within 24 hours after treatment. I was wondering if you have applied doxycyclin to iPSCs and know what...
27 January 2022 1,411 1 View
Dear all, I am planning to study histology and want to do that at home. I have only stained a blood smear with Giemsa but now want to try a wider range of animal tissues. What reagents will I...
25 February 2021 5,673 3 View
Dear all, I am trying to harvest the low-expression plasmids in the mentioned E.coli strain. I constantly gel very low concentrations (30-50 ng/uL while the concentration is usually around 100...
09 February 2021 4,374 4 View
Dear all, I am propagating some plasmids with a human gene and an ampicillin resistance gene. My logic is that if ampicillin concentration will be increased, the concentration of plasmid (pSF)...
15 January 2021 914 3 View
Dear all, I was doing site-directed mutagenesis with one mutagenesis primer (fwd) and one normal primer (rev) to amplify a 6.5-kb plasmid. Then KLD was performed with dnp1 destroying the original...
11 January 2021 1,117 6 View
Hi everyone, I have a box with vials of sterile mRNA in nuclease-free water. I keep it at -80C freezer but I accidentally left it on the bench at room temperature for more than 3 days before...
01 January 1970 8,202 3 View
Dear colleagues, I ran a colony PCR to see if colonies contain the insert after the transformaiton with a Gibson assembly. The products are ~1810 bp (ORF+primer-added additional 300 bp). When I...
01 January 1970 5,731 1 View
*Long post* Dear colleagues, I am trying to linearise a plasmid fragment of ~6.5kb size by PCR with primers that generate overhangs to then perform a Gibson assembly. It is done with a standard Q5...
01 January 1970 6,105 5 View
Dear colleagues, I am synthesising some RNA which contains the IRES, ORF, 5' UTR and 3'UTR.The original DNA ORF will be replaced with a new ORF with Gibson reaction. Although, there will be a...
01 January 1970 6,556 2 View
Dear colleagues, I transformed DH5alpha with 70 ng or pUC-based construct per protocol (C2987 NEB). There were not many colonies seen next day (approximately 20 tiny colonies after 1:10 dilution...
01 January 1970 5,493 3 View
Hi all, What are some nanoparticles that can safely carry mRNA without degrading it? I suspect that zeolitic imidazolate fraework (ZIF-8 and UiO-66) NPs break down eGFP mRNA as no HEK293T and...
01 January 1970 1,867 5 View
Dear colleagues, I made some mRNA product of 910 bp, from a restriction-enzyme-linearised pUC-derived vector. Standard NEB IVT T7 cleancap synthesis kit protocol was followed (E2080 NEB) as...
01 January 1970 7,584 1 View
Hi everyone, I added 3 separate modifications to the eGFP mRNA to see how they would affect protein yield. (e.g. modif.1, modif.2 and modif.3, n=3 replicates per modificaiton). I looked at eGFP...
01 January 1970 4,335 2 View
Hi everyone, I am about to perform electroporation (Neon) of NK-92 cells with eGFP mRNA. I was wondering why is electroporation efficiency so low in NK cells (40-70%) and survival (~50%) after...
01 January 1970 4,665 3 View
Dear all, What could these things be in a clean mammalian cell media? Yeast? Mycoplasma colonies? Media components sedimenting? They are as big or a little bigger than lymphocyte cells. No...
01 January 1970 2,288 5 View
Dear all, May someone please share a protocol on how to make chemically-competent E.coli DH5-alpha cells? I have a few vials of NEB C2987H highly-competent E.coli DH5a cells and need to make more...
01 January 1970 7,256 3 View
Dear all, Modern cancer treatments and immunotherapies costs can easily top $1,000,000 per patient because of cumbersome, expensive manufacturing and patents. The success rate of, say, CAR-T cell...
01 January 1970 7,711 2 View
Hi everyone, I will soon start PhD and discussed areas of research with my supervisor (hepatology, cancer and mRNA). I read on these areas, but cannot really come up with a project design, or,...
01 January 1970 691 8 View
Dear all, Can you please suggest recommendations on how to do a deep clean of the shaker incubator (Eppendorf New Brunswick S41i)? There is mold growing on the sticky pads, walls, grid and other...
01 January 1970 7,656 3 View
Dear colleagues, Do you know how to store an aqueous soluion of folic acid? (The SDS sheet says it is inherently unstable) I resuspended it in water with adjusting pH to ~10 with NaOH until all...
01 January 1970 8,964 2 View
Hi everyone, What statistical test would be most appropriate for this experiment? I want to compare two conditions against one another at different time points, e.g. mRNA1 vs mRNA2 at 24 hours,...
01 January 1970 3,058 5 View
Hi everyone, I have 1.5 mL eppendorf tubes with nanoparticle pellets in water (ZIF8, UIO-66 and UIO-66-PEG). No matter how hard I try to homogenise them into single nanoparticles, using pipetting...
01 January 1970 782 3 View
Hi all, I cannot resuspend nanoparticles (ZIF-8, UiO-66 and UiO-66 Peg) in water without them immediately forming a pellet and sinking. I tried pipetting up and down and vortexing for a few...
01 January 1970 7,035 2 View
Dear colleagues, I am writing a paper on non-specific antibody binding to self-antigens. Do you know whether maturing B-cells are exposed to all self-antigen peptides possible (trillions or...
01 January 1970 5,641 2 View
