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[QUESTION] Isolating one individual HEK293 cell from T75 flask culture?

  • I have been growing HEK293 cells, splitting to maintain healthy confluency

  • I will transfect these cells with a specific plasmid that expresses GFP

  • I need to isolate only cells that have taken up the plasmid

How can I do this with only GFP as an indicator (the plasmid does not contain antibiotic resistance except for Kanamycin, which I don't believe is compatible with HEK293), I have been told that cloning rings may be a good way but I do not fully understand the procedure.

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2 comments
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Isolating one individual HEK293 cell from T75 flask.

  • I have been growing HEK293 cells, splitting to maintain healthy confluency
  • I will transfect these cells with a specific plasmid that expresses GFP
  • I need to isolate only cells that have taken up the plasmid

How can I do this with only GFP as an indicator (the plasmid does not contain antibiotic resistance except for Kanamycin, which I don't believe is compatible with HEK293)> I have been told that cloning rings may be a good way but I do not fully understand the procedure.

4
14 comments

Genuine question: How did u wear that without succumbing to the sun's flames?

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Original Poster3 points · 9 days ago

My suit is self-cooling (serious).

Are you the Batman with a Batman lambo?

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Original Poster3 points · 9 days ago

I wish - if I was rich I wouldn’t be doing charity work lol

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I'm a Canadian studying in the U.S. legally with a student VISA. I find this absolutely absurd - things such as voting for government should be reserved solely for citizens.

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Question about Protein Structure Positioning in CHIMERA

Say I have a protein structure consisting of multiple alpha-helices. How do I select one alpha-helix and position the entire protein so that this one alpha helix is perfectly vertical, or horizontal?

The purpose is for comparative analysis between two very similar protein structures, I want to vertically align a helix that is identical in both structures so that the areas of non-homology can be easily seen and outlined in the images.

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2 comments
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Chimera Software Question: Protein Structure Positioning?

Say I have a protein structure consisting of multiple alpha-helices. How do I select one alpha-helix and position the entire protein so that this one alpha helix is perfectly vertical, or horizontal?

The purpose is for comparative analysis between two very similar protein structures, I want to vertically align a helix that is identical in both structures so that the areas of non-homology can be easily seen and outlined in the images.

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2 comments
4

Question: CHIMERA Protein Structure Positioning?

Say I have a protein structure consisting of multiple alpha-helices. How do I select one alpha-helix and position the entire protein so that this one alpha helix is perfectly vertical, or horizontal?

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5 comments

Relative to what? Proteins in an aqueous fluid do not remain in a single orientation relative to the "floor".

Theoretically if you know the total protein structure, you can attach an antibody to a plate that binds a certain part of the protein (not necessarily the specific helix itself) which resuls in the specific helix being horizontal or vertical to the plate bottom...I am failing to see a knowledge gain from doing such a thing though.

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Original Poster1 point · 1 month ago

The purpose is for comparative analysis between two very similar protein structures, I want to vertically align a helix that is identical in both structures so that the areas of non-homology can be easily seen and outlined in images.

If the structures are already solved could you throw it in a 3d program and do it that way? If that doesn't sound like what you mean/ you want specific recommendations r/biochemistry or r/labrats may be more help.

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Original Poster1 point · 1 month ago

That is what I am using CHIMERA for, as stated in my title. But you're totally right, there has to be some specialists on those subs who have an answer.

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Question: CHIMERA: How do I open multiple PDB files in separate windows (Mac OS X).

I'm running Chimera on my mac. When I have a protein structure from a PDB file open and running, and I try to open a 2nd one it just overlaps the two structures in one window/session. I would like to view the two structures separately in two separate windows.

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8 comments

Glad that worked. Chimera's never really been my favourite - much more so a fan of VMD.

To move one structure and not the other: Open both in the same window. Go to Favorite -> model panel. Then untick one of the molecules under the "active" column. You should now be able to move the one by clicking the mouse wheel. Once you make them both active again they will rotate and whatnot together.

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Original Poster1 point · 1 month ago

Awesome thank you. Since we're on the topic of Chimera... When I click Tools > Depiction > Color Secondary Structure, I have the ability to visualize Helix, Strand, and Coil.

Let's say I want to visualize a hinge region or a turn, any way I can do that?

It colours based on a program called dssp or something (I forget what it specifically uses). To custom colour stuff, just hold down ctrl and then click parts of the structure with your mouse, then actions -> color

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Original Poster1 point · 1 month ago

I have a follow-up question regarding positioning of a protein in chimera.

Say I have a protein structure consisting of multiple alpha-helices. How do I select one alpha-helix and position the entire protein so that this one alpha helix is perfectly vertical, or horizontal?

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What kind of bears living your area of the world?

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Original Poster3 points · 1 month ago

Well I do gene-therapy research, so they are all microscopic :)

[deleted]
3 points · 1 month ago

Is .02 inches microscopic? Why do I feel like they're smaller than 0.02 in?

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Original Poster1 point · 1 month ago

I don't work with tardigrades specifically, but yes you would need a microscope to visualize this little organism properly - with the naked eye it would just look like a dot. From Googling it seems they can range in size from 0.002 to 0.05 inches.

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Original Poster2 points · 1 month ago

Happy to help! :)

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Here’s some advice, you just wrote an excellent outline for a book. And not some run of the mill self-help book, instead you came up with something that contains useful and practical advice. If I were you I would put this into a simple easy to read book format and watch yourself make some money.

Original Poster1 point · 1 month ago

Thanks, I'll honestly consider that. Are there any areas you can suggest that are missing?

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Well one thing I’ve found worth thinking about:

Productivity = efficiency + effectiveness

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New reader hear, anyone know of some good books along the theme of Spartacus (the TV series)?

I just finished Spartacus (the TV series) and am craving for more along the same theme. Other productions I enjoyed were Troy (2004), The Eagle (2011), Gladiator (2000), 300 (2006), Centurion (2010), etc.

What are some good books with similar themes? Or even based on the same characters.

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5 comments

You might like The Song of Achilles by Madeline Miller

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Original Poster2 points · 1 month ago

Sounds intriguing! Thank you!

Gates of Fire-same battle as 300, but the real story.

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Original Poster1 point · 1 month ago

Excellent, will check it out!

20 points · 1 month ago

I get the article

Being productive is great but if you were inefficient in the process, you might have lost more time than needed.

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As stated in previous comments...

productivity = efficiency + effectiveness

The definitions I use to clarify things:

  • Effective is accomplishing what is intended.
  • Efficient is accomplishing something/anything with the fewest resources (faster, cheaper, less effort, etc.).
  • Productive is being efficient at being effective. Or, accomplishing what is intended with the fewest resources.

So, you can be effective without being efficient, and you can be efficient without being effective, but you can't be productive unless you are both effective and efficient.

Also somewhere in those words, it makes sense to me that 'effective' includes making choices about purposeful, meaningful goals, objectives, and an overarching mission.

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What this person here said, is the best definition.

productivity = efficient effectiveness, as defined above.

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Help needed finding some plasmid prep protocols.

My lab just received our own custom plasmid that we ordered in the form of a dry large scale prep (a small tube with just dry DNA in it). First thing i did was wrap the tube in aluminum foil and put it in the fridge.

Protocols:

1) I need a protocol for resuspending the plasmid DNA for both use and long term storage (I believe we need TE buffer for this?).

2) I need a protocol for transfection of HEK293 cells (mammalian) with our plasmid. Our cells are adherent type, growing in a media of DMEM+FBS+Glutamax.

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10 comments
3 points · 1 month ago · edited 1 month ago

+1 for all of this. I usually resuspend my plasmids to a volume of 40ng/ul (ie 4ug of lyophilized plasmid + 100ul TE). Be sure to centrifuge the tube first to make sure no dry DNA is on the cap. After that I transform into NEB Stable E. coli with the high efficiency protocol.

I transfect with the calcium phosphate method but all the cool kids use lipofectamine these days so I'll let you make your choice there

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Original Poster1 point · 1 month ago

Interesting, I have both lipofectamine and CaPhosphate at my disposal.

What stresses me out the most though is should I first resuspend ALL of the dry DNA (I have 50μg) into 1X TE buffer? If so at what amount (you said you typically use 100μL TE for 4μg DNA)?

In my case that would convert to 1.25mL of TE Buffer added to 50μg of DNA. Do I need sterile distilled water as well?

2 points · 1 month ago · edited 1 month ago

I resuspend everything. I use 40ng/ul out of convenience, you are free to do what you want. For example, if you add 1ml TE, you now have 50ng/ul. Consider this your frozen stock. I'm not sure if you will be going back to this a lot, but if so, maybe you can make a few 50ul (again, arbitrary volume) aliquots for the sake of not freeze/thawing a million times.

Do you have 1x TE at your disposal or will you have to make it? If you're making it yourself, then yes you'll need some ddH2O.

I've never done lipofectamine transfections before, I started with calcium phosphate back in November and have been doing it since I never learned anything else. I get good results with it (for my purposes) and thought it was pretty easy to pick up.

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Original Poster1 point · 1 month ago

Aha, I am beginning to understand better now.

If I add 1mL TE to my 50μg of DNA, I would have a concentration of 50μg/mL.

Now let's say out of this 50μg/mL DNA TE Buffer solution I want to make 10 aliquots ... Do i just add 10mL TE Buffer and distribute everything into 10 separate tubes?

(I know that sounds really uneducated, but I want to make sure I don't screw up)

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Help needed finding some plasmid prep protocols.

My lab just received our own custom plasmid that we ordered in the form of a dry large scale prep (a small tube with just dry DNA in it). First thing i did was wrap the tube in alluminum foil and stick it in the fridge.

Protocols: 1) I need a protocol for resuspending the plasmid DNA for both use and long term storage (I believe we need TE buffer for this?). 2) I need a protocol for transfection of HEK293 cells (mammalian) with our plasmid. Our cells are adherent type, growing in a media of DMEM+FBS+Glutamax.

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3 comments
  • Either TE or elution buffer from plasmid prep kits work for resuspension and storing of plasmid DNA. It's good practice to make 100X stock of your plasmid and aliquot it into tubes to be stored at -20C. Although plasmids are fairly sturdy, you still want to avoid too many cycles of freeze/thaw.
    • Before resuspension, allow the tube to reach room temperature; don't surprise your cold DNA with warm buffer!
    • Use sterile tips and work in a cleaner area of the lab while resuspending. It's worth to crack open a new box of commercial sterile pipette tips for precious material such as your custom plasmid.
  • Check your stock concentration after resuspension — you might have to dilute it — using a NanoDrop or other spectrophotometric methods to help you keep your transfections consistent.
  • Another good practice is to transform a fresh batch of DH5alpha or other competent cells with your plasmid for future use, especially since custom plasmid orders are somewhat pricy. The transformed cells should be stored at -80C.
  • You're in luck! HEK293 cells are readily transfected with high yields, so you won't need as much DNA or liposome in the process. Too much liposome can make cells sick and they're also pricy. I've used Lipofectamine 2000 and 3000 in the past and they both worked very well with HEK293 transfection. As I said, I used less DNA than recommended in the protocols and things still worked out, but if you're new, stick to the protocol to start and optimize later.

All the best!

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Original Poster1 point · 1 month ago

Thank you very much for writing that out!

Technical question:

Assuming I have 50μg of dried DNA, I also have 1X TE Buffer, is there a step by step by step protocol of how I can prepare the stock?

Secondly, to prepare the DNA for transfection, I'm assuming I simply create a batch of DNA in sterile distilled water at the concentration listed in the transfection protocol, and then follow the rest of the protocol for transfection (including lipofectamine if listed). But is that it? Just the DNA dissolved into sterile distilled water?

11 points · 1 month ago

I don't believe that specific dogs go to heaven, though I believe that there will be dogs in the new creation.

But, I can't find scriptural support for my first statement just as someone who believes the opposite can't find scriptural support for the opposite position. It's all conjecture.

Either way, heaven will be you in the presence of Jesus. That will be better than any dog, no matter how much you love them.

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I like that you admitted the lack of scriptural support no matter where you stand with this specific issue. I would sustain that God is love and expresses grace far greater than judgement and condemnation. In this sense, the love one might experience for a dog may even be a mere taste of Christ’s love in the form of said dog. Not that the dog is Christ lol, but that we may feel the love of Christ through the dog. If one so desperately wishes their dog to be with them in heaven, one may end up being graciously surprised and satisfied with what God has prepared for us there!

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NOOB HELP: makeblastdb won't run in terminal (Mac OS X)

So I downloaded the BLAST+ executables from NCBI (from here: ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/).

I opened terminal, copied and pasted the path to where I unzipped the BLAST+ file, and when I try to use makeblastdb I get "command not found"

Here is a screenshot (just blotted out my username): https://i.imgur.com/QEMsr1Q.jpg

I don't understand why it's not working, the path is correct, all files are there, I downloaded the 64bit version to match my mac.

The reason I'm doing this is because I want to make a local database out of a FASTA file to run a BLAST against. If anyone has any easier way of doing this please share, as my code proficiency is not strong.

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2 comments

Try "./makeblastdb"

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Original Poster1 point · 1 month ago

Worked like a charm, with one line of a reply you just helped me generate possibly invaluable data for my project. Thank you dearly!

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Locating a gene of interest within a set of contigs from a transcriptome sequence.

Excuse my lack of understanding.

So I have the sequence of a gene of interest I want to locate and compare within the transcriptome of a certain organism (P. leucopus).

The only resource I could find online is here: https://datadryad.org/resource/doi:10.5061/dryad.6hc0f/3

From my understanding the FASTA file from this resource contains a number of contigs.

As I mentioned before I have the sequence of my gene of interest. Now I want to find where it is located within this group data of contigs, so that I can compare my sequence with the one that should be within the transcriptome sequence.

My logic is that this would be similar to CTRL+F function when searching for a phrase within a large document of text.

Where do I begin? What software should i use? Do I open up each contig manually and search for my sequence?

Any help would be greatly appreciated!

Excuse my lack of understanding, I am new to the field of bioinformatics.

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4 comments

You can BLAST your query sequence against your contigs. Just make a BLAST database from your contig file.

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Original Poster2 points · 2 months ago

Very good advice, I didn't think of using NCBI blast! thank you.

You are an underappreciated genius and I salute you.

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And you, Neo, you are the one.

BUT, you have to admit that That's good advice coming from a 13 year old

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Assuming the 13 year old subject is human.

Good insult coming from a bunch of mops

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Aye, you used me Skinner! Ya used me!

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NCBI Blast Question. Please help a noob (screenshot attached)

I ran a blast using the DNA sequence for Human FOXP3 as the query sequence against the search set "nucleotide collection" database for the organism mus musculus. Being a noob, I would like to know specifically what the variables I circled in red in the screenshot mean. Max Score; Total Score; Query Cover; E value; Ident; Accession.

I apologize in advance for my lack of knowledge, I would be grateful for any help!

Screenshot: https://imgur.com/a/fvGiguw

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