[DIYbio] Re: Four questions about reliability of a wet lab action

I think the answer to Q1 is that it can be highly variable depending on user, but if you're using a known transformation system, i.e common host/chassis, vector and insert then the turn around can be a couple of days if everything lines up i.e primers/PCRs work, transformation is reasonably efficient etc. There's often waiting times for some of the steps where you can be doing other things.

Q2 transformation efficiency (I'm assuming this is what you mean by producing new germ line) is something you can measure thoughout the process. If you're using a well characterised system there may be historical data on this. My experience in a DIY setting is efficiencies can be lower for various reasons.

Q3 I think you mean false positives? Depends on how you're testing for and what you consider success. If you've selected for an insert on the plate, and tested for the right size of insert (maybe through colony PCR) you've probably narrowed it down a lot. If you get that sequenced and it's exactly the right thing with base calls of high confidence, you're probably doing well.

Q4 If you're intending for the gene product to be expressed there's further characterisation you can do.  e.g protein chromatography or metabolite analysis. Do you intend to isolate the product, e.g nickel chelation with a poly-His sequence, and do you isolate a product of expected size on SDS-PAGE is a common approach.

On Wednesday, August 31, 2022 at 2:40:46 PM UTC+1 dank...@gmail.com wrote:

                        

Greeting reader(s):

I posted this question buried in a deeper strand, I think maybe it has to bubble up to get noticed enough to snag opinions, answers; maybe some direct experience 'stories':

Question began 'sort of' to to Sean Sullivan, possibly but really a general question

Here are four questions about creating a gene edit and expression, ex Yeast, even e coli, etc... Some plate and Petri dish wetlab style work. All are about framing expectations and resources, the assumption is the technology is 'best we can get', and/or almost isn't the point, anyway.

Reference Situation:

*) Gene BP count is 3K NT, promoter, tails, introns if any etc known to work generally.

*) Expression system known, like locally managed Yeast or 'whatever'

*) Selection itself is reliable, UV florescence or some other property

Questions:

Q1) How many labor hours is typical, no paperwork time, just wet lab including end game verification ?

Q2) Whats the success rate of a new germ line emerging in percent ?

Q3) How often is a success rate wrong ultimately. not that the Gene did do what is expected, but some 'other' flaw made it not really get expressed ?

Q4) Is sequencing the believed usable final modified colony invariably reliable enough to make Q3 irrelevant ?

Is Q2) Closer to what ? 30% 80 %

For Q1) I mean hands on time with endless revisits to the plates/wells, dishes, tubes, etc. and so on, not including incubation times, centerfuge, etc.

Thanks in advance of anyone who answers. As people like me want to make this technology, not science, this is a big thing hugely.

Thanks!

Daniel B Kolis

my ref: nafl, 31 Aug 2022, diybio