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DevGrp's Content

There have been 20 items by DevGrp (Search limited from 08-April 19)


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#10904 Alternative stains for DNA

Posted by DevGrp on 09 February 2005 - 02:17 AM in Molecular Biology

How about Sybrsafe from Invitrogen, Its "safer" and seems as sensitive. It also has the advantage of being compatible with normal UV tranilumiators and is that expensive. If you work out the cost compared to EtBr its really expensive but if you look at the other costs in a gel -agarose, markers, samples, time- its insignificant. The LIST PRICE (who pays those?) in the UK is 34 / 40 x 100ml gels

I'm busy trying to convert my insitute over to Sybrsafe.

https://catalog.invi...u...e gel stain



#9892 How to cut cost in a molecular biolog lab?

Posted by DevGrp on 11 January 2005 - 03:52 AM in Build and Run a Lab

Your lab could be different, but here in the UK unless we are doing REALLY expensive experiments I have found that often money saving ideas tend to waste time and that the biggest bill is actually wages. Work out how much the staff cost per day and then decide how effecive your cost saving measures are against that (I was surprized).

That said I have found that looking at the sources of your consumerables and changing surpliers can save lots of money. Contact the local reps and push them abit, they usually have some room to reduce their prices.
good luck.
I have also been in the position when grants run out of bartering consumerables with other labs.



#9868 Plasmid DNA storage

Posted by DevGrp on 10 January 2005 - 09:00 AM in Molecular Biology

The problem with self defrosting freezers is that they cycle their temperature (heating up cooling down ) to avoid frosting up. This can have the effect of continually defrosting / refreezing your samples. They can also dry out samples in a similar way to freeze drying



#9554 Rapid screening of recombinants

Posted by DevGrp on 23 December 2004 - 01:55 AM in Molecular Biology

I would do 50+ 10ul PCR reactions, for us thats cheaper than the technicians / my time to do 50+ mini-preps (and the restriction enzymes for digests can be expensive) and if you do the colony PCR in the morning you can have positive colonies by the afternoon and even save a day.

The other alternative to screen "lots" of colonies is to do plate lifts onto membranes and then do hybridization but that can get expensive (32P / non radioactive detection)
good luck



#9536 breast cancer cell primary culture

Posted by DevGrp on 22 December 2004 - 01:10 AM in Cell Biology

Either I could email you our method or a similar method can be found in Pubmed paper

good luck


Ian



#9524 Rapid screening of recombinants

Posted by DevGrp on 21 December 2004 - 08:42 AM in Molecular Biology

Yep I've always used colony PCR



#9505 breast cancer cell primary culture

Posted by DevGrp on 20 December 2004 - 07:38 AM in Cell Biology

I used to do breast primary culture. I'll see if I can find the protocol tonight amoung my old backups or in Electronic copies of my students thesis. Otherwise our group published it in:

Speirs V., Green A. R., Walton D. S., Kerin M. J., Carleton P. J., Fox J. N., Desai S. B. and Atkin S. L., Short-term primary culture of epithelial cells derived from human breast tumours. Br. J. Cancer,78(11):1421-9 1998

V. Speirs, A. R. Green and M. C. White, Collagenase III: a superior enzyme for complete disaggregation and improved viability of normal and malignant breast tissue. In Vitro Cell. Dev. Biol. 32 (1996), pp. 7274.

J. T. Emerman and D. A. Wilkinson, Routine culturing of normal, dysplastic and malignant human mammary epithelial cells from small tissue samples. In Vitro Cell. Dev. Biol. 26 (1990), pp. 11861194.

I've just checked and none of these seem to be online. can you get them from a library?



#9472 hep-2 & Hep-G2 cell line

Posted by DevGrp on 17 December 2004 - 02:45 AM in Cell Biology

Some primary cells need it, some don't. We used gelatin for HUVECS but nothing for human primary breast or endometrial epithelial cells.



#9448 hep-2 & Hep-G2 cell line

Posted by DevGrp on 16 December 2004 - 03:24 AM in Cell Biology

By coated do you mean something like sarstedts cell+ (yellowcap) "for adherent cells"?

I've mainly used the standard sarstedt (red cap) flasks for cell culture and had a lot of problems growing HepG2s.

Think you might just have solved it.

Pity I that was two years ago!

Thanks

Ian



#9332 ORF-open reading frame??

Posted by DevGrp on 10 December 2004 - 06:29 AM in Molecular Biology

Sorry, I could see that confusion in the definitions I got from the website

"The "Open" part refers to the lack of a stop codon WITHIN the reading frame"

Important word is "within"
open reading frame: long run of codons starting with a start ending in a stop with no stops in the middle.



#9288 ECL Western blotting detection kit

Posted by DevGrp on 09 December 2004 - 01:46 AM in Protein and Proteomics

Well in therory any sensitive CCD camera with an appropriate filter and in some kind of dark room / dark box should work.
The only problem would be getting software to allow you to collect the image over several minutes (5-15min)



#9268 ECL Western blotting detection kit

Posted by DevGrp on 08 December 2004 - 08:59 AM in Protein and Proteomics

If by light box you mean something like the Epichemi II darkroom (UVP Lab Products, UK) which has a CCD camera with appropriate filters or Biorads VersaDoc system then the answer is yes. I actually found the Epichem more sensitive that film



#9262 why so many small inserts?

Posted by DevGrp on 08 December 2004 - 06:07 AM in Molecular Biology

Two questions:
Does your PCR also give some primer dimers?
Did you run your PCR product on a gel and then cut out the band you want, purify it and then cloned it?


I have experenced similar problems and put it down to cloning the primer dimers / small unwanted PCR products. Gel purifying the band you actually want to clone first. solved it for me.

Hope this helps
Ian



#9259 ORF-open reading frame??

Posted by DevGrp on 08 December 2004 - 05:29 AM in Molecular Biology

Medicine.net

Open reading frame: A long sequence of DNA that has no stop codon (no signal to stop reading) and therefore may encode part or all of a protein.


The "Open" part refers to the lack of a stop codon within the reading frame.

In practical terms when I get a DNA sequence which may encode a protein I run it through some software called vector NTi. This searches for ORFs in the 6 possible (3 forward/3 reverse) triplet frames. The default is that an ORF should start with a start codon (ATG or GTG) go on for at least 300 codons (900bp) and end in a stop codon (TAA TGA TAG). Usually 5 of the frames wont have long enough ORFs (ie closed reading frames, start codons with stops shortly afterwards) and only one of the frames will produce an ORF.



#8800 Need help with results of transformation...

Posted by DevGrp on 22 November 2004 - 02:56 AM in Microbiology

You said that the contaminants were mold . Ampicillin doesn't effect the growth of molds and is actually isolated from the filamentous mold Penicillium chrysogenum .



#8769 E. coli culture contamination

Posted by DevGrp on 19 November 2004 - 09:26 AM in Microbiology

Sounds like Staphylococcus aureus to me. I'd streak your E.coli out, get a single white colony and start again. You may have preped a mixed E.coli / staph culture which would explain your low plasmid yeild.



#8764 Salmon sperm DNA

Posted by DevGrp on 19 November 2004 - 09:05 AM in Molecular Biology

I must admit when doing southerns I've frequently left it out without any major background problems. It's usually added to fill up the membrane to whcih DNA hasn't already bound, so that your probe doesn't stick to the membrane in a non-specific way (dired milk / BSA / gelatin is used for a similar reason in westerns)
I have found using Denharts or Church pre-hybridisation buffer without salmon (or herring) sperm DNA works just as well.
Ian



#8761 How many cell passages to be safe ?

Posted by DevGrp on 19 November 2004 - 08:15 AM in Cell Biology

A couple of years ago I got an undergraduate student working on endothelial cell receptor expression to look at the changes in expression of 3 receptors over 25 passages of EAHY926 endothelial cells. The expression of ERbeta dropped by 300 fold, IcamI by 250 fold and GR by 8 fold (qRT-PCR). This seemed to explain why a previous student couldn't detect any ERbeta in her EAHY cells which had been high passage.
After this we always went back to the early passage stocks every 15 passages.
I think different cells behave in different ways. always keep an eye out for changes in the phenotype you are studying.



#8689 How to prepare Sephadex microcentrifuge columns?

Posted by DevGrp on 17 November 2004 - 06:07 AM in Molecular Biology

You can make rough and ready ones using a 600ul microfuge tube inside a 1.5 / 2 ml tube. Put a small whole in the bottom of the 600ul tube with a hot wire and then put some glass wool inside the tube to stop the sephadex escaping. You can then add your sephadex in solution and this gives you a mini-spin column. Experiment with the size of whole / amount of glass wool / amount of sephadex.
Sorry I can't give any more details. It was 6-7 years ago in a different lab
Hope this helps
Ian



#8600 why is LDH secreted

Posted by DevGrp on 12 November 2004 - 03:01 AM in Molecular Biology

LDH is released from cells with damaged / destroyed cell membranes during cell death. An LDH kit made by Roche is a standard way to look at cell membrane damage / cell necrosis

Hydrogen peroxide (in the cells I have worked on) will at low concentrations cause apopotosis while at higher concentrations it causes necrosis.
For a nice graph showing this see figure 1 J Endocrinology. 1999 May;161(2):199-210. Functional inactivation of the oestrogen receptor by the antioestrogen, ZM 182780, sensitises tumour cells to reactive oxygen species. Newton CJ, Drummond N, Burgoyne CH, Speirs V, Stalla GK, Atkin SL.

I can email you the PDF if you can't access it.

I expect you are inducing oxidative stress which is causing cell death. Have a look at your cells under the microscope you may be able to cell apoptosis / necrosis.

I was interested that you had also detected an increase in LDH transcription after H2O2 treatment. This I never measured and would have to think about.

Hope this helps

Ian




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