34 Questions 18 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 1,258 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 3,241 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 1,420 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 5,754 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 4,400 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 938 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 1,143 6 View
Good afternoon, I have stored P2 and N3 buffers in ice for about one hour. The P2 buffer turned milky and I discarded it. The N3 buffer didn't seem to have any chemical changes, so I am wondering...
25 December 2020 5,998 0 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 8,211 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 5,740 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 6,116 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 6,567 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 5,503 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 1,879 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 7,594 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 4,346 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 4,678 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 2,298 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 7,268 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 7,722 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 705 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 7,666 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 8,976 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 3,070 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 794 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 7,046 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 5,651 2 View
Hi everyone, I have DNA templates for T7 IVT mRNA transcription that are between 250 and 1050 bp long. I want to run T7 IVT mRNA synthesis using NEB2080 kit as recommended, but since tempates are...
01 January 1970 1,163 0 View
Hi everyone, I am looking for a ribosome complex on an mRNA .PDB file to visualise the interaction in the PyMol. I was so far unsuccessful at finding it in PDB or Uniprot databases, but want to...
01 January 1970 614 2 View
Dear colleagues, I am wondering whether the size of an individual e.coli colony matters as to how much plasmid it produces? Would larger colonies give higher plasmid yield, or would smaller...
01 January 1970 5,484 3 View
Hi everyone, I ran a 1% agarose in 1X TAE with SybrSafe. I ran same mRNA (left well: undenatured, right well: denatured). The only difference is that right band mRNA was denatured at 72'C for 3...
01 January 1970 3,728 3 View
Dear everyone, May you please recommend a protocol for electroporation of NK-92 cells using Neon machine? I have never done that before and woul dbe happy for recommendations if anyone had...
01 January 1970 1,829 0 View
Hi everyone, Why could the band appear smudged? I have amplified a 300 bp DNA G-block using Q5 2X mastermix (NEB) for 34 cycles, using 10-uM primers and 1 ng of template G-block as per protocol...
01 January 1970 1,250 4 View
I am looking for a mammalian cell media with a few specific supplements, but it is not available for purchase from any vendor. The lab-grade supplements are pretty expensive and are sold in either...
01 January 1970 1,193 3 View
