Custom protein labeling & conjugation service provides personalized solutions designed to supply researchers with the widest choice of labels and conjugates to meet their specific needs . We offers high quality, efficient and cost-saving labeling & conjugation services with the largest selections of labels and protocols. We can help you decide on the most suitable conjugate or approach for your applications and optimize labeling procedures toward your specific need. We will find a solution for you at Creative BioMart .

http://www.creativeb...in-Labeling.htm

There are also lot of inhibitors for signal pathway, but you should find the exact inhibitor by the target by yourself.

http://www.creative-enzymes.com/cate/Enzyme-Inhibitors_72.html

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.

http://www.creative-peptides.com/services/custom-peptide-synthesis.html

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.

http://www.creativeb...Interaction.htm

There are some information on lentviral vectors, maybe useful.

http://www.creative-biogene.com/Product/Premade-Lentivirus-Particles-list-85-1.html

I don't why this happened. When I did qPCR, it seems had similar picture, but had no influence for the last result.

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?

Gel purify after digestion.

 

 

Besides, did you run agrose gel to test whether the plasmid digested completely?

I do not see any relationship between DNA and peptides from your condition, how did you get such conclusion?

I saw DNA loading buffer from Thermo contains 1% SDS, which could eliminate DNA-protein interactions, prevent appearance of additional bands due to annealing of DNA molecules with cohesive ends.

I think it is impossible to clean the green dye by that way, you'd better use new polymerase. 

I encountered this before. I chose several clone with same plasmid, but some of them would grow slower than other, that will also influence the amount of plasmid.

 Did you try another clone, did it have same situation?

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.

Could you provide your gene sequence? It will be helpful for us to analysis.

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.

I never encounter this situation before, did you check your template of the 5' homology region before?

How much time did you run your reaction?  It is perhaps your reaction time not long enough?

Did dNTP enough for the retrotranscription?

Did you run DNA gel to test your production,how about your result?

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.

http://www.creativeb...search_gp36.htm

There are also instruction about protein A， but it seem not related to HIV, you can take as reference.

http://www.creativeb...h_Protein A.htm

This time depends on the time strain needed to grow. Protocol could be use for most situation, but  you may adjust according to the specific circumstances.

I saw some kits could measure the metal, but not the total metal concentration, just for specific one. For example, 

http://www.creativeb...search_Iron.htm

http://www.creativeb...-Kit-462630.htm

This is really a very big question. Usually we use gene-knockout technology to knock-out the gene, or use inhibitor/siRNA to inhibit the gene expression for short time. So what is your specific goals for your experiment?

Maybe you could ask your boss directly, and what he want you to do with that protein powder.

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.

I'm sorry I cannot tell you the difference these two. Usually I use Lipo2000 for plasmids, but Lipo2000 works well with shRNA, too.