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1

Historically speaking, the use of VSVG (Vesicular Stomatitis Virus G protein) labeled with GFP was indeed a practical method to define the secretory pathway. (@tyersome comment) However, I am concerned that by the time everything is set up, the cell will be at steady state, which means GFP signal would be present throughout the secretory pathway, preventing ...


0

While it's difficult to know just what is going wrong in somebody else's laboratory, apparently paradoxical effects like these are unfortunately common in laboratory work. Assuming that these are indeed well-established inhibitors that really should be working, let's consider why they might not be in your hands. The mechanisms of molecular interaction are ...


2

There is a degree of variability in milk, and in some cases this cause problems in protein work. Despite being a standard for ELISAs, it's not known to be the most reliable blocking buffer. As far as cryoprotection goes, variability doesn't seem to be a major concern. Standardization varies depending on manufacturer. The Sigma link you included reports a ...


13

Purely going off experience here having used golden gate assembly methods for 5+ years now, there is a definite lack of literature regarding small part assemblies. In my current lab, we use CIDAR MoClo (a golden gate method) and have assembled 100's of constructs successfully with parts as small as ~ 70 bp. However, when we assemble smaller parts we tend to ...


9

As you may have realized, crystal violet can be replaced by a lot of dyes since the ethanol will wash out the dye from the gram negative cells. Methylene blue is a nice choice - malachite green may also work. A weak concentration of hydrochloric acid (3%) is also a possible replacement for the ethanol. A 3% HCl solution is used in the acid-fast stain but it ...


3

The answer depends on the application and level of oversight/compliance you require. Most research labs I've seen use de-ionized or even reverse-osmosis water (from a tap) for most culture media applications. But when I was doing trace-metal analysis, I had to use higher grade water for almost all steps in my experiment. Mostly, we needed a certification ...


0

The OP is dealing with combinatorial complexity, too. So they might want to look at this figure if they are mutagenizing at the second position of the codon. Again, from Wikipedia (Genetic Code)


0

CyberDope was first written at MIT, then again at Kairos, then again by me (privately) in Mathematica. The program gives the best "dopes" for a target set of amino acids. You will need someone who has Mathematica to view this program in ".nb" format and to run it. I would further suggest they upgrade the program into Mathematica ...


1

This answer only concerns Streptococcus pneumoniae. The lab I worked in used cheap ingredients, the same as everyone else claimed to in this field. The only shocking thing I found was that the sterile media (already prepared) that I was given to use for DNA extraction did not work (DNA would be fragmented). It turned out the media had been waiting in the ...


4

It looks like Merck have different levels of quality control for their products. MQ 100 being the lowest up to MQ 600 being the highest. From what I can see the MQ200 product here has defined composition (of salt, tryptone and yeast extract in g/L) whereas the cheaper MQ100 product does not appear to have its composition ensured. I am sure your cultures will ...


10

This sort of method is indeed quite useful and frequently used in synthetic biology: I've used a similar approach before to generate 5' insulators for promoters. Calculating the exact theoretical likelihood of what you want, so far as I understand, is a complex combinatoric equation that I wasn't able to get a nice closed form on with a few minutes of work. ...


8

I don't have hard data to share regarding you question, but I can provide some anecdotal evidence. I have used golden gate assembly to build hundreds of plasmids using ~20bp annealed oligos, as well as hundreds of plasmids using ~70bp promoter+RBS, 720 bp eGFP, and ~70 bp terminators encoded on plasmids into a ~6kb backbone. In these cases I use 40 fmol ...


5

It may also be worth considering how you are amplifying and isolating your small part. Gel extraction kits sometimes have an optimum DNA size ranges that may prevent recovery. Or if your part is similar to your primer length you may see considerable contamination in your 'purified' product if say the size of a single RBS.


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