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8

To summarize from Hanahan's paper (see below for reference), he carried out a systematic review of conditions necessary to improve transfection efficiency. Among the conditions tested were the presence of various cations (Ca2+, Cs+, Mn2+, K+, Mg2+), DMSO, and growth temperatures (and a few other conditions not so commonly used these days). He worked with a ...


7

ATryn is a human antithrombin produced in the milk of transgenic goats by GTC Biotherapeutics. It has FDA approval and I believe that it is available for prescription in the USA. Added later, after the emphasis of the question changed somewhat. Proteins produced in a mammalian system are more likely to have post-translational modifications that are much ...


6

(Super Optimal broth with Catabolite Repression) SOC media has glucose where as Luria Broth (LB) doesn't. SOB also has a more delicate salt balance. Naturally the glucose will help the cells recover and grow faster after the heat shock. Your transformation efficiency should improve since less cells will die. On the other hand, the reason why SOC media ...


6

The rationale for the choice of higher organisms as the producing source is based on costs and biological activity. Biological activity. In their active forms, various proteins have post-translational modifications (i.e. glycosylation) which are difficult to reproduce in bacteria. Alan's answer is already exhaustive. Costs. Mammalian cell lines are easier ...


6

There is no problem to transform bacteria with more than one plasmid. The usual methods (either chemical competent cells or electroporation) are working fine, although electroporation might give you better results, as the cells take up more DNA. On the downside you have to make sure that your cells are prepared nicely, otherwise the electroporation cell will ...


4

If you are interested in the history of molecular biology this is an interesting question. Basically transformation came to be used to describe experiments in which the phenotype of an organism was changed by the uptake of DNA, and because of the way this developed in bacterial systems this DNA was usually a plasmid. Then it became possible to use ...


4

After looking around for a bit, I came across a few potential solutions however they are not a definitive answer to your question and requires testing under your experimental conditions to see which works best. This page contained one excellent suggestion and that is to select for cells that do not clump during the passage, by pooling the cells in 15ml of ...


4

I have never worked much with yeast, but I can still give some answers: Salmon sperm is used as a the so called "carrier DNA". It is thought to bind to the yeast cell wall and thus prevents that the DNA which shall be transformed does so. This raises the transformation efficiency. See here for more details: "Transformation of yeast by lithium ...


3

In the research lab? I thought about it for a while and found no usage. But it could be interesting for clinical applications to prevent bacteria spreading their resistance genes (which are usually organized on extra chromosomal plasmids) and thus preventing multi-resistant bacteria.


3

I have done that and it works without any problems. We harvested the cultures at the optimal timepoint (density) and then froze them to do maxipreps later on. Therefore we simply thawed the culture, made sure it didn't got too warm (meaning we worked on ice) and then simply followed the maxiprep protocol. No problems with yield or quality of the plasmide. ...


2

http://www.protocol-online.org/biology-forums/posts/26634.html I found this link - it says 'we don't really know'. It says PEG binds DNA, I assume shielding the membrane from its negative charge and allowing internalization to happen. I would guess that the amphipathic nature of PEG, being partly hydrophobic, also helps soften up the membrane. ...


2

Electroporation is a fairly common way to introduce exogenous nucleic acids into cells. Its name essentially describes the process - an electric voltage potential is applied across a biological membrane, eventually leading to the production of conducting hydrophilic pores. The image below is from the Wikipedia page linked above, showing non-conducting ...


1

Heat shock transformation alters membrane fluidity creating pores: A sudden increase in temperature creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell. Reference: Journal of Visualized Experiments. Bacterial Transformation: The Heat Shock Method. 2014. ...


1

I did try another transformation: again with our electrocompetent cells Cells form other lab chemically competent cells I made The good thing is that on chemically competent cells there is no weir colonies, however efficiency of transformation was very low, but still, I think it is ok - will confirm it in next day if I really got the right insert. On ...


1

If you want to express them in an operon, make sure you have an RBS at the 5' end of each coding sequence. If you place one 10bp downstream without it having an RBS, it will be transcribed, but not translated... If you want more help, paste the DNA code of each as a comment.


1

Here is a report that shows significant increase in transformation efficiency for large plasmids (transformation was by electroporation. However the paper has been cited fewer than ten times so clearly the technique did not catch on. Zhixing, Y and Nahon, J-L (1995) DNA gyrase improves DNA transformation of E.coli cells with large recombinant plasmids. ...


1

Different bacteria have different modes of transformation and as I understand it is Haemophilus influenzae that takes up the single strand whereas ?Streptococcus [i am not sure which bacteria it is] takes up dsDNA. I intuitively think that DNAses are not that common like RNAses. Moreover, natural competence is associated with stress and is even triggered ...


1

According to this site, for yeast (I assume this means Saccharomyces cerevisiae) you should use 50 µg/ml to 200 µg/ml; for fungi (!) use 100 µg/ml to 300 µg/ml. The site also stresses the importance of the pH of the medium. I think that, unless a Hansenula expert comes along, you will have to try an initial experiment to measure the sensitivity of ...


1

In my experience the elution buffer does not matter (both for E.coli and Mycobacteria). I have heard this before from co-workers, but it usually is based on superstition or anecdotal evidence. I have compared water/EB elution and never saw a difference in sparking (in fact, I usually get slightly more colonies with EB). When I get arcing (sparks), it's ...



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