Molecular Biology

Using cancer cells to fight cancer is not a bad idea, (its like fighting fire with fire) but identification of cells as malignant in early stages of cancer is itself a challenging duty. Cancer cells have specific markers that can be used to identify them and can be distinguished from the healthy ones, but our immune system may create a hindrance as inclusion of any cancer cells from outside will be treated as foreign antigen. So the experiment can be started using nude mice or immunocompromised mice and later tested in humans.

The salt and the alcohol can affect the DNA and the combinations are better for specific situations. If you have access to Maniatis (now Sambrook and Russell) there is a good overview of information in there to help you decide the best precipitation method for your situation.

Hi, if smear is coming in your PCR product, you may need to increase the annealing temperature or decrease the magnesium (if you added Mg yourself and it wasn’t already in the mix.) You may also be adding too much template. Just work with three parameter, I am sure you won’t get any smear further. Good luck.

Good luck! :)

30% Glycerol could be standard

This ia a very common problem, when your DNA concentration is low (which is often the case after a gel extraction). The reason for this is that you have to add a large amount (Vol) of your insert (and vector). After a gel extrcation, the DNA is not as pure as you might think-in fact it is quite salty and this salt intereferres with the ligation buffer and the ligase wont work that well (they are bitches anyway :-). But if you have many colonies for your only vector control ligation, it seems your vector is not cut well or re-ligates. Always cut over night and add Alk. phospatase in the morning for like 4h or so (directly to the restriction reaction, ignore the buffer for the Alk. phos,) then run it on the gel and extract the vector from the gel. You should have only very few colonies for your control reaction now, but this will not solve your problem, for the ligation you have several options to improve, best to do all of them: 1, increase your DNA concentration, vector and insert (e.g. grow 3x as big cultures, run 3 PCRs or digests and pool together in a small volume) then you have to add only a little bit to the ligation reaction. 2. Don't trust a nanodrop, when your concentration is <50ng/ml. better load a few microliters to a gel and compare the band intensity with the marker (which should have a known amount/concentration) 2. Once you have a high concentration of DNA, you might want to desalt both the vector and the insert before adding it to the ligation, which will purify it even more. I use sephadex for desalting but there are many options. 3.. Always do a 20ul ligation reaction (or bigger) even if you don’t need it, because this will dilute the contamination from your DNA as well (insert and vector). 4. Ligate overnight-I normally do it at RT, 16°C, 8°C and 4°C never worked for me…but it depends I guess. 5. desalt your ligation before transformation (if you don’t and your insert DNA is contaminated with salts this will also cause the transformation to work better in the control ligation than in the actual cloning ligation) Cheers!

Primer blast works pretty well. I always double check primer binding to specific exons or introns on http://uswest.ensembl.org/index.html.

I am not doing the old fashion ligation of vector and fragment anymore but use only the In-Fusion HD cloning system form Clontec (cat no 639645). It works like a charm and you can cut down on supplies by using only half of it or even less for the fusion reaction and transformation. Plate about 20-30 uL of the transformation/SOC/bacteria mix and you will end up with a large amount of colonies.

For a global analysis before focusing on the most altered thanscription factor

yea..that seems fine...i was wondering if I could use SPR for this.any suggestions?

Here is a link that will help to adjust according to the temperature ! http://www.encorbio.com/protocols/AM-SO4.htm

VectorNTI is very good. Unfortunately is not free. You can do lots of stuff in Bioedit but do not know if you can get a circular genome with it.

You can try this with the polymerases mentioned by others, but it is dependent on the secondary structures which can be formed with the denatured DNA during the annealing phase. Alternatively, if you know your DNA region, you should prepare several primers to get overlapping PCR products.

-Find your sequence by Ensemble, -Design primers by Primer 3 that is perfect, easy to use, and recommended by Pubmed. -You can also check mismatches and dimers by Oligo Analyzer.

Thanks Reidunn Aalen. But I was no thinking to form an octamer of purely H2B histones. I was wondering if, despite there is no interaction between the H2B histones, is possible that DNA can be wrapped around the H2B histones.

Bead beating might be required to completely lyse the cells.

Since the place is just 1 he away transfer at ambient temperature won't be a problem.keeping in contact with ice may give freeze-thaw effect which isn't advisable.Stab or a slant would be ok.You may keep a spare transfered culture at ur lab.Best of luck.

You should check out the www.addgene.database. You can call them if you can't find what you need. In particular this part of the site might be helpful: http://www.addgene.org/empty_backbones/

I think the best to use the media, to which cells were adopted before without antibiotic

As far as I know, most study suggest the production of QS signal molecules are growth-phase dependent, peaking at the later mid-exponential phase. However, to measure/evaluate the exact concentration dynamics is much complex. the secretion of signal molecules is either related to the cell density or influenced by medium. more intriguingly, the indirect assay of signal molecules is restrained by multiple factors, e.g the luminescent assay of AI-2 is inhibited by both acid and glucose, while stimulated by boron. A more objective method may be examining their (or their chemical modified derivants) concentrations at different time points. Regarding the uptake of signal molecules by bacteria, some strains are proposed to utilize them as carbon source when sugars are in short supply, commonly after the stationary phase. To be frankly, if one of any question posted by you can be answered, a nice paper is completed.

I have stored my cDNA at 4 degrees for 3 weeks and it worked all the time.

I had a similar problem once. In the end, my protein was just not as soluble (even thought it had been small and should have no complex secondary structure-but maybe it needs other proteins to fold properly). Anyway, I found that I fell out once I applied it to the Ni column-the way I check this was with the bradford reagent. Basically you add the fresh ni-column slurry to the bradford and nothing should happen....if your protein stays stuck in the ni-column slurry, and you add this to the breadford reagent it turns blue....simple test, needs 10minutes or so-I believe worth a try. If this is the case you wil have to change the buffer to make your protein stay sable during the chromatography-and remember every protein is different, if something works with one, it does not have to work with another

There are several in EU funded consortia, which you may find on the CORDIS website: http://cordis.europa.eu/newsearch/index.cfm?page=advSearch

If you are sending vital cells frozen in DMSO, I would ship them on dry ice by a special overnight courier service such as DHL or Fedex. Use a styrofoam box and sufficient dry ice and include a written statement such as: This package contains XX vials each containing XX ml of XX. The contents are non-infectious, non-contagious, non-hazardous without any commercial value; and for research purposes only

I think the major area of interest is the way in which chromatin, DNA methylation and non-coding RNAs cooperate to control gene expression and cell phenotype.

We had detected P-Glycoprotein and ABCG2 in same cancer cell line. "Blebbishields, the Emergency Program for Cancer Stem Cells: Sphere Formation and Tumorigenesis after Apoptosis" Goodwin Jinesh G, Woonyoung Choi, Jay B. Shah, Eugene K. Lee, Daniel Levi Willis, Ashish M. Kamat https://www.researchgate.net/publication/233742776_Blebbishields_the_Emergency_Program_for_Cancer_Stem_Cells_Sphere_Formation_and_Tumorigenesis_after_Apoptosis