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10

I think that cells get damaged at -20°C not because they are stored for a long period of time, but because they undergo cycles of thawing and freezing (the ice crystals that form damage them). I never keep cells at -20°C. I store them at -80°C in 50% glycerol. The idea behind the glycerol is that it serves as cryoprotecor. It forms hydrogen bonds with the ...


8

Let's say that we have gene A, followed by gene B so that they overlap with 100 bases. If you want to knock out only gene A, you can do the following: PCR gene A and clone it into a vector PCR gene B and clone it into another vector Insert a antibiotic resistance marker at the overlapping region of gene A and B now you don't have functional gene A and ...


8

Gergana covered the "why" part of your question. +1 If all you have at the moment is a -20°C and mostly what you want to store is E. coli harboring plasmids, I'd recommend preparing plasmid DNA and storing that at -20°C. The DNA will stay stable in the medium term, and you can re-transform into E. coli once you've got your -80°C freezer up and running. And ...


8

The minimum requirement for E. coli and other bacteria to grow and survive is called minimal medium. It's even defined at Merriam-Webster: a medium that contains only inorganic salts, a simple carbon source (as carbon dioxide or glucose), and water Water and glucose are pretty easy, but the source of salts may often change; regardless, you really need ...


7

This is particularly an issue when you do maxipreps and the pellet is 200x the size of the pellet from a miniprep. The reasons why? Maybe you're pelleting at too high of a g-force (pure speculation). Alternatively, you could be growing the cells too long. I typically miniprep after a 12 hr culture. If you're seeing any darker colors in your pellet, you have ...


7

To be honest, you really shouldn't be buying such things if you're not prepared to handle them properly. If you work with a proper lab, you will be inindated with vendors trying to sell you these and many others. But to answer your question, if you are looking to purchase many such items ATCC is an excellent resource. Not only will you have to meet ...


5

The cited article describes effects of Tris on the outer membrane of E. coli. Like all Gram negatives E. coli has an inner (or cytoplasmic) membrane which is a typical phospholipid bilayer membrane containing many membrane proteins including transporters and, of course, an electron transport chain. Surrounding this there is a layer of peptidoglycan, and then ...


5

I believe Addgene send strains as bacterial stabs. This is the protocol from Qiagen. E. coli strains can also be stored for up to 1 year as stabs in soft agar. Stab cultures are used to transport or send bacterial strains to other labs. Prepare stab cultures as follows: Prepare and autoclave LB agar (standard LB medium containing 0.7% agar) ...


5

For any kind of frozen cell stock (for cell culture or bacteria) we routinely use freezing vials similar to these: http://www.sigmaaldrich.com/catalog/ProductDetail.do?D7=0&N5=SEARCH_CONCAT_PNO%7CBRAND_KEY&N4=V9255%7CSIGMA&N25=0&QS=ON&F=SPEC These tubes are internally threaded with a rubber grommet to seal the lid and make the tube air ...


5

the devil is in the details here - the logic is okay, but there is no experiment here. Too many unanswered questions. Are you doing anything specific to see optimize RT activity? How are you preventing your GFP mRNA from being turned into protein, producing much larger signal than you will ever see from RT activity? Once its integrated into the ...


5

This must depend upon the conditions in question, but I think it would not be very long. The length of a generation for e coli can be 12 minutes or 24 hours, so that gives some idea of a typical time. I did find this interesting case in the literature. They found that even as you subject e coli to minimal nutrients they are expressing genes that prepare ...


4

I'm with @Mad Scientist. Save yourself the trouble and do this in an in vitro system. At the moment, you don't even know if the HIV-RT will express or will be active in E .coli. Rather, express mutated HIV-RT in T-Cells or Rabbit Lysate. Purify. Add in a RNA template with the appropriate primer. Hydrolyze your RNA Detect your cDNA.


4

The answers already posted about glycerol being a cryoprotectant and ice crystals are correct. The other thing to consider is that enzymes still retain some activity at -20°C and though the reaction rate is slower, the cells will degrade until they are no longer viable. I always store glycerol stocks of bacteria at -80°C. Many common E. Coli strains ...


4

What I do to avoid this is to ressuspend the cells by pipetting up and down the P1 (ressuspension) buffer on the side of the tube. This way, every time takes only a bit of cells every time and big clumps are avoided. The only times I used to get big clumps hard to ressuspend were when I went poking at the pellets first.


4

Some things to consider. If you spinning too fast and too long, that is going to pack the pellet more. You can spin longer at a slower speed and you will notice your pellet is not as tightly packed. I always do 2000 rpm, 20 minutes, 4C if I'm doing a midi or maxi sized prep and harvesting bacteria in a 50ml conical. If I'm doing a miniprep I will take the ...


4

We routinely use E. coli preps from 16-18h overnight cultures. The cultures don't show any overt signs of lysis (sometimes you can see cellular debris if the culture has indeed overgrown). I have also noticed inconsistent times when resuspending the pellet. For my own use, centrifuging at too great a g-force or for too long can increase the likelihood of ...


4

E. coli is mostly harmless; only a few strains are harmful. I don't believe the route by which gut biota is established has been entirely established for any species but, for example, koala feed their faeces to their offspring to help them establish biota capable of digesting eucalyptus. It seems that a small proportion of ingested bacteria somehow survive ...


4

Let me first clarify the difference between anaerobic respiration and fermentation. Literally respiration refers to breathing, but its definition has been extended to include cellular metabolism that leads to ATP generation. Fermentation, as you said depends on substrate level phosphorylation. Anaerobic respiration on the other hand just means ATP ...


4

NAD+ is important in this step, since it is co-factor for the glyceraldehyde-3-phosphate dehydrogenase (G3PDH), which acts as a acceptor for the hydrogen atom from the C1 (see below). If you look at the reaction, the aldehyde from the C1 is oxidized to a carboxylic acid which in a second step is turned into a phosphoester. To do so, a cysteine from the ...


4

This question got me thinking about what are the metabolic enzymes that take oxygen up in E.coli. I searched the metacyc database for reactions that consume molecular oxygen and there are only 3 that take in oxygen and one that produces oxygen. All three consumers of oxygen in E.coli are the oxidation of ubiquinone by at two sites in cytochrome-bcl or by ...


3

Expression of sfiA (sulA) causes filamentation during the SOS response, so presumably sfiA is induced, as part of SOS, in response to some sort of DNA damage. Once the DNA damage is repaired the SOS genes, including sfiA, will be repressed again and normal growth will resume.


3

Not sure that I have ever come across trace metals in recipes (I assume that you aren't referring to the Ca/Mg salts component of M9), but it is certainly the case that many K12 strains (e.g. C600, DH5α) require thiamine because they are thi mutants. This doesn't apply to BL21 since it is from a different lineage (it is a B strain). In a lab where ...


3

I would rinse the cells off the agar using a small volume of LB or similar rich medium. Then use a small aliquot (10 μl) to inoculate a 5 ml culture under selection to see if anything will grow. Meanwhile the remainder of the cell suspension from the vial can be used as input for your favourite miniprep procedure. If necessary, the DNA that you recover ...


3

I like that you are considering a simple and standardized approach. However, there are a number of potential pitfalls to consider with this proposal. First of all, what specific RT activity are you trying to quantify? Is the the transcription speed or accuracy, or are you trying to characterize something else entirely? If you are measuring speed, how and ...


3

Yes, but no. In other words, this quote is not probably not true in the ways you'd think. Bacteria can survive on practically nothing for long periods of time, but whether you call that life is subjective. Nitrogen is necessary for all the co-enzymes and proteins to sustain life. In order to get energy, if E coli. needs to metabolize nitrogen to waste at ...


3

The plasma membrane is quite permeable to oxygen and thus oxygen enters the cell simply by diffusion. Reactive oxygen species can be reduced enzymatically in aerobic organisms. Obligate anaerobes lack or don't produce sufficient quantities of these enzymes. An organism that doesn't use oxygen for metabolism but is also not relatively harmed by it can be ...


3

It is not uncommon for cells to have parallel pathways for same outcome. This ensures foolproof response and makes the system robust. E.coli also has another sensor for aerotaxis (Aer and Tsr proteins). See my answer on your previous post and the linked paper. Also look for coherent feed forward network motifs.


2

Over time I suspect that ice crystals will form in the colonies from defrost cycles in the freezer and the moisture in the agar. This will cause rupture of the bacteria membranes and over time may destroy the bulk of the bacteria cells. The plates will also desiccate over time as well from the defrosting cycles of the freezer. You may be able to leave ...


2

I think the best way is to use lyophilization. The American Type Culture Collection send the strains that way.


2

I found that E. coli exposed to predators such as amoeba changes completely from normal form to filaments (on agar) that look wound up like spaghetti but are not only longer but significantly wider. In this form the predator can't attack E. coli and it lives among the amoeba for extended periods. It does not look like a simple point mutation to me, which ...



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