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Andrea Fortina's Content
There have been 55 items by Andrea Fortina (Search limited from 22-April 20)
Hi, I used to order this service from the company below. I thought it is OK for me. But it was not from Shanghai, I don't know whether it is convenient for you.
Y2H, Phage display technology, Surface Plasmon Resonance (SPR, Fluorescence Resonance Energy Transfer (FRET), Protein Array, Co-Immunoprecipitation (CO-IP), Pull-downs, Biomolecular fluorescence complementation (BiFC), Far-western Blotting
All these method coule be used for protein interaction. Some of them you could do in your own lab directly, and can also find a company for time saving.
Actually the first thing that pops my mind is that the transformation is working just fine and there is nothing wrong.
Is it possible that something wrong with the competent cells? Maybe the cells get contaminated during the process making the competent cells?
For most standard cloning (where the insert is smaller than the vector) a 3 insert : 1 vector ratio will work just fine. But for your reaction, your insert concentration is much lower than the vector. So you could try only use 0.1-0.2ul vector and use the rest of the volume for the insert. Use 10* buffer, do not add water.
Agreed the amount of tris and EDTA is usually negligible for most downstream applications.
Agree too. I also elute my DNA in water. And stored at -20C. But I seldom use them for stored so long, although it could keep ligation efficiency. I usually use fresh materials.
I never use puromycin for bacteria, if you want to select bacteria obtain some plasmid, maybe you could check whether the plasmid has other selection marker. Generally, shuttle plasmid has two selectable marker at least, one for bacteria and one for cell.
HIV-1 and HIV-2 appear to package their RNA differently. HIV-1 binds to any appropriate RNA whereas HIV-2 preferentially binds to mRNA which creates the Gag protein itself. This means that HIV-1 is better able to mutate. HIV-2 is transmitted in the same ways as HIV-1: Through exposure to bodily fluids such as blood, semen, tears and vaginal fluids. Immunodeficiency develops more slowly with HIV-2.
HIV-2 is less infectious in the early stages of the virus
han with HIV-1. The infectiousness of HIV-2 increases as the virus progresses. Major differences include reduced pathogenicity of HIV-2 relative to HIV-1, enhanced immune control of HIV-2 infection and often some degree of CD4-independence. Despite considerable sequence and phenotypic differences between HIV-1 and 2 envelopes, structurally they are quite similar. Both membrane-anchored proteins eventually form the 6-helix bundles from the N-terminal and C-terminal regions of the ectodomain, which is common to many viral and cellular fusion proteins and which seems to drive fusion.
HIV-1 gp41 helical regions can form more stable 6-helix bundles than HIV-2 gp41 helical regions however HIV-2 fusion occurs at a lower threshold temperature (25°C), does not require Ca2+ in the medium, is insensitive to treatment of target cells with cytochalasin B, and is not affected by target membrane glycosphingolipid composition.
There are also instruction about protein A， but it seem not related to HIV, you can take as reference.
Do you want to aliquot your solution? If not, that's not worth to do it. Something could reuse in lab, some containers (including tubes) I used for cell experiment, could be washed and autoclave sterilized, and then be used for bacterial culture or experiments outside the cell room.