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11

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

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

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

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

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 ...


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 ...


6

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 ...


6

There are some databases in which you can search for E.coli gene expression data: GenExpDB: E. coli Gene Expression Database Many Microbe Microarrays Database (M3D): A resource of microbial gene expression data Stanford MicroArray Database (use the search tool to find relevant organisms) Colombos (COLlection Of Microarrays for Bacterial OrganismS) ...


6

I've found a nice review that has many details on plasmid replication in general, and several papers about pSC101 in detail, and I'll try to extract the key information from these papers. First of all your plasmid as an ori region that contains so called iteron: In many cases, the origin of replication contains directly repeated sequences, termed ...


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

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 ...


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

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 1....


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

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

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 ...


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 ...


4

One is not normally required to serially dilute E. coli cultures for spectrophotometric measurements, at least in the experiments where the OD value is important. For most protein expression work, the consensus is to start the induction at OD 0.6, and for most chemically competent cells, the optimal OD ranges from 0.3 to 0.8 depending on the competency ...


4

Its simple: Mass = Density * Volume = 10^3 (Kg/m3) * (10^-18 m3) = 10^-15 Kg which is equal to 10^-12 gram or 1pico gram


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

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

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 ...



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