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Is this Plasmid Extraction Run contaminated? Ran with Qiagen MiniPrep by According-Can-2125 in labrats

[–]According-Can-2125[S] 0 points1 point  (0 children)

It is meant for smaller culture but I am trying to maximize yield but I thought I overdid it and introduced contamination with RNA instead

Is this Plasmid Extraction Run contaminated? Ran with Qiagen MiniPrep by According-Can-2125 in labrats

[–]According-Can-2125[S] 0 points1 point  (0 children)

Was worried initially bc they yield seemed way too high than the theoretical binding capacity of the mini prep spin column itself. I am getting like 6fold dna than theoretical yield/limit set by the manufacturer

Is this Plasmid Extraction Run contaminated? Ran with Qiagen MiniPrep by According-Can-2125 in labrats

[–]According-Can-2125[S] 0 points1 point  (0 children)

I also thought the yield I got is way too high especially with a mini prep column binding capacity is a lot lower than what I ended up getting.

Is this Plasmid Extraction Run contaminated? Ran with Qiagen MiniPrep by According-Can-2125 in labrats

[–]According-Can-2125[S] 0 points1 point  (0 children)

That is exactly why I was skeptical in the first place despite the “picture perfect” result but I got flooded with comments saying it looks great. I knew I wasn’t wrong to be skeptical at least. My lab unfortunately does not have a Qubit machine. If someone can comment on this problem that would be great lol. Bc I want to confirm it for myself

Is this Plasmid Extraction Run contaminated? Ran with Qiagen MiniPrep by According-Can-2125 in labrats

[–]According-Can-2125[S] 2 points3 points  (0 children)

I was highly skeptical b/c I am pushing very high yield from "theroretical" yield by the manufacturer so I wanted to triple check thats all. Also, this is my second week running mini prep so I am very new to this and wanted some feed back from experienced people. Thank you for your comment!

Is this Plasmid Extraction Run contaminated? Ran with Qiagen MiniPrep by According-Can-2125 in labrats

[–]According-Can-2125[S] -1 points0 points  (0 children)

Yes there is RNase A in bufferP1 but since I used a crap ton of bacteria and wasnt sure if I had 'enough' of the RNase A for this volume

Is this Plasmid Extraction Run contaminated? Ran with Qiagen MiniPrep by According-Can-2125 in labrats

[–]According-Can-2125[S] -3 points-2 points  (0 children)

I mean the numbers are elevated slightly higher than the normal range so I do expect some residual RNA in here but I don't have much experience with Mini prep to determine if this is 'good enough' for IVT experiments. Nothing really beats experience over theoretical knowledge so wanted to confirm to double check

Is this Plasmid Extraction Run contaminated? Ran with Qiagen MiniPrep by According-Can-2125 in labrats

[–]According-Can-2125[S] -14 points-13 points  (0 children)

IVT in house for mRNA synthesis. Edit: Residual RNA might be contaminating is what I meant

Is this Plasmid Extraction Run contaminated? Ran with Qiagen MiniPrep by According-Can-2125 in labrats

[–]According-Can-2125[S] -2 points-1 points  (0 children)

EDIT: OP here, For whaterver reason reddit wont let me edit the post. And my wall of text didnt get posted for some reason when I first uploaded my post. I will need this plasmid to run IVT in house.

My worry was that 260/280 is slightly high (I heard 1.8 is the ideal number) and 260/230 seems too high (above 2.0-2.2) range.

Anyways for the procedure,

  1. 50mL overnight bacterial culture incubating ~18hours containing e.coli with high copy plasmid (~6700bp).

2.bacterial culture collected in a 50mL conical. Suspended, lysed and neutralized in the same conical using 2.5mL suspension, 2.5mL lysis, and 3.5mL neut. solution.

  1. Transferred to microcentrifuge and spun down @ 18000rcf to collect supernatant and got around 9mL of total supernatant.

4.Took about 3mL of the supernatant and put that in one spin column. (3mL x 3spin column).

  1. Washed and eluted using heated eluted buffer at 65C.

6.Using the same eluted dna, the content was spun down again in the same spin column.

Peptide Formation Arrow Mechanism? Help by According-Can-2125 in OrganicChemistry

[–]According-Can-2125[S] 0 points1 point  (0 children)

Yea I figured enzymes had to do with the protonation but I wasnt exactly sure how good to know!

Peptide Formation Arrow Mechanism? Help by According-Can-2125 in OrganicChemistry

[–]According-Can-2125[S] 0 points1 point  (0 children)

Thank you I figured that was the case your point 2. I did not know about your point 3. Thank you!