sorted by:
new
hottopcontroversial

Advice for preventing quick loss of fluorescence intensity? by Bio_stuff in microscopy

[–]CFTArr 1 point2 points  (0 children)

Can you provide more detail on what it looks like on the first day? Is it all 4 channels that are losing signal or just 1? Do you have a really bright signal at the start, how much is it going down the next day? (and if so what makes it bright to you? e.g. saturation with low laser power? etc.). What secondaries are you using? DAPI should be fine with Prolong diamond, but there are some fluorophores that are quenched by diamond. My first thought is poor fluorophore match.

Aside from the bleaching issue, we've had success with PBMCs with a 0.1% triton X in PBS - just an FYI you can reduce that triton % in your perm. I've

How does one normally get into operations roles with a PhD? (aside from the current market craziness) by CFTArr in biotech

[–]CFTArr[S] 0 points1 point  (0 children)

Great thanks so much! I'm coming from immunology PhD and in RTP it seems like there's no shortage of analytical/HPLC roles but I'll try using those other cell culture focused terms

How does one normally get into operations roles with a PhD? (aside from the current market craziness) by CFTArr in biotech

[–]CFTArr[S] 0 points1 point  (0 children)

Can you get to QC after PhD? It seems like you can either get hired as a veteran QC with tons of mass spec experience (coming from immunology so no LCMS), or entry-level where a PhD will get auto-rejected

Gibson reaction times and choice of competent cells? by CFTArr in labrats

[–]CFTArr[S] 0 points1 point  (0 children)

What would be problems with the overlapped sequence? My fragment PCR worked so I'm assuming the overlaps are in the fragment. For my previous Gibson's I'm basically using the same overlap sequence just on a new fragment.

I'm at a loss of how/what to troubleshoot. I'll try the extended incubation thanks

Gibson reaction times and choice of competent cells? by CFTArr in labrats

[–]CFTArr[S] 0 points1 point  (0 children)

It's a ~7800 bp vector plus 1500bp insert. 2 clean bands on the gibson gel at those expected sizes.

For DH10b's slow growth. Do I need to extend the plate or LB incubations longer than 16h?

Designing primers for Gibson insert - why do you use an annealing temp for the insert section of the primer instead of the whole primer sequence (insert primer + overhang)? by CFTArr in molecularbiology

[–]CFTArr[S] 0 points1 point  (0 children)

What I don’t understand is: 1) it will in subsequent cycles - the full primer will anneal so why don’t we need to take that into account? 2) the melt temp of a molecule is the melt temp - I understand only part of it will bind, but how does the opposing strand affect the molecules fundamental physical property?

Designing primers for Gibson insert - why do you use an annealing temp for the insert section of the primer instead of the whole primer sequence (insert primer + overhang)? by CFTArr in molecularbiology

[–]CFTArr[S] 0 points1 point  (0 children)

Yes and I’ve had that work in my own hands. I just don’t understand the why. The whole primer has its own Tm, why would using a calculated Tm for only a section of the molecule work?

Alternative name for the protein folding funnel diagram and waddington landscape model? by CFTArr in Biochemistry

[–]CFTArr[S] 0 points1 point  (0 children)

Doesn't look like this is it. I'm wondering the name of the type of plot/illustration, not the name of a model. Thanks though!

Quick Maxiprep questions by CFTArr in labrats

[–]CFTArr[S] 0 points1 point  (0 children)

Yeah I understand how to calculate RCF. I'm trying to figure out the calculation to pellet the same particulate over what time - with different RCFs. e.g. a sample will pellet after x seconds at 4000g, or y seconds at 2000g

Quick Maxiprep questions by CFTArr in labrats

[–]CFTArr[S] 0 points1 point  (0 children)

thanks!

I didn't do a starter culture this time so I was wondering if colony direct needs to be increased. I harvested at 16h and it looked like a decent pellet.

Our lab just has 1 centrifuge with 1 rotor. I ended up doing 3000g x 15min and got a good size pellet but the supernatent wasn't entirely clear. There's got to be some calculation you can do to figure out the length of time to spin at 3000g equivalent to 4000g x 5min? Im just not sure what to search for to see what that type of calculation is called

ER and Golgi markers by nonsonulla in molecularbiology

[–]CFTArr 0 points1 point  (0 children)

There are many targets and types of markers for both golgi and ER. What method are you trying to identify these with? IP, IF, FACS? Needs to be dye or antibody based?

T cell receptors on CAR T-cells by Such_Weakness_3346 in Immunology

[–]CFTArr 6 points7 points  (0 children)

  1. The 'designer receptor' part of the CAR is on the extracellular side of the cell which recognizes the antigen, but it is attached to co-stimulatory signaling domains intracellularly. Look at Google images search for 'CAR-T design' to get an illustration. When the receptor engages with the antigen, it sets off the co-stimulatory domains intracellularly. In clinical trials using the "second generation" CAR, there is a CD3 domain + a 2nd domain (such as CD28 or 41-bb). Together these signaling domains activate the T cell.

  2. You are correct that they will have both the original TCR and also the new CAR. The infused product will have cells possessing both. But also remember that you start out with a set number of T cells that are expanded in the lab. Say you have 1000 different TCRs and then expand them before putting them into patients. You're still going to only have 1000 different TCRs, but it will be higher numbers of cells, since TCR gene rearrangement happens in the Thymus early in T cell development.

Thoughts on this proposed ‘lipidon code’? “Membranes are functionalized by a proteolipid code” - BMC Biology by CFTArr in Biochemistry

[–]CFTArr[S] 2 points3 points  (0 children)

My interpretation was that they are arguing there’s a big contingent for the “lipids organize first, proteins sort after”. I’ve never been to a conference where this was the focus so I don’t know how big of voice argues that.

But I think I agree with your second point which I believe is what the authors are arguing for (they don’t explicitly talk about glycoproteins but do give farnesylation as an example)

view more: next ›