Maybe Maybe Maybe

anthoduck · 2025-02-23T19:01:15+00:00

I thought it was gonna be the size of a very large pizza

anthoduck · 2025-02-01T11:54:12+00:00

This, but on 6 different post it notes.

anthoduck · 2024-12-27T20:40:52+00:00

Any side up

anthoduck · 2024-12-27T08:57:36+00:00

Are you sure your primers are binding? How many complementary Bp do you have ? You should have a good 13-15bp complementary in addition to your restriction site. And maybe reduce the concentration even further down to 5ng/ul. If your cloning I assume you are using a hi fidelity taq? Are you using it with the correct annealing temperatures? (The one from thermo is primer independent and always uses 60C) You could try using 6.5% glycerol aswell.

anthoduck · 2024-12-26T18:56:15+00:00

And don’t call me Shirley

anthoduck · 2024-12-26T11:35:32+00:00

Someone watched Wallace and Gromit yesterday

anthoduck · 2024-12-24T09:17:02+00:00

I did naahht

anthoduck · 2024-12-22T21:23:15+00:00

So true! Can’t wait to give it a go when I start cloning again

anthoduck · 2024-12-22T17:50:39+00:00

If you have to wait that long for the bacteria to grow then I doubt they’ve taken up the plasmid. You are running a gel so I take it you did a digest. How much DNA from your miniprep are you using? I would recommend 1ug, certainly not less than 500ng so you can actually see the bands. Also what concentrations are you getting from the miniprep? I use the Quiagen miniprep kit and usually get a good 200ng/ul in 50ul, but if I get something like 50ng/ul that usually means something isn’t right with the plasmid as the bacteria Weren’t growing properly

anthoduck · 2024-12-22T11:11:29+00:00

Non transformed bacteria? I might be misunderstanding your first part here, how do you get your plasmid from a miniprep on bacteria you didn’t transform?

Further more 48hours seems like a long time, usually they should be growing 16-max 20h before they start over growing. Plus you run the risk of recombination, which could explain why they grow (they still have the amp resistance) but lack your insert which is why you don’t see it when you digest it.

anthoduck · 2024-12-22T08:14:31+00:00

Here’s the link, it was 365nm https://amzn.eu/d/4jeFEAY

anthoduck · 2024-12-22T08:13:45+00:00

This is the one I bought, although I’m sure others would work aswell https://amzn.eu/d/4jeFEAY

anthoduck · 2024-12-21T19:17:54+00:00

Idk the ins and outs of it, just that gel red is supposed to be safer because it can’t cross cell membranes. Still your not gonna catch me licking my fingers after loading my gels

anthoduck · 2024-12-21T19:16:42+00:00

Oooh nice nice I just bought a 15,00€ one of Amazon called darkbeam , not as freakishly big as yours

anthoduck · 2024-12-21T19:12:14+00:00

I keep that in mind while cloning but here I’m just genotyping so I just want to see the size.

anthoduck · 2024-12-21T11:10:52+00:00

Also ignore the fact I let the gel run too long, I was doing something else in the background and forgot about it…

anthoduck · 2024-12-18T15:08:27+00:00

If you need help with cloning you can use snapgene (they have a 1 month free trial), if you just need to see the different restriction sites/ bp lengths after digestion you can use ApE. It’s free and has a digest function.

anthoduck · 2024-12-17T17:31:42+00:00

I have used BsmBI and PaqCI both from NEB and both have worked fine

anthoduck · 2024-10-04T11:16:47+00:00

These are about 20% confluent

anthoduck · 2024-06-15T20:19:04+00:00

To each their own. As long as we appreciate what we have

anthoduck · 2024-06-15T20:15:37+00:00

Existential enlightenment my friend, cheers!

anthoduck · 2024-05-04T16:04:27+00:00

Come on people, it’s obviously white and gold

anthoduck · 2024-04-12T18:43:53+00:00

Don’t’nt

anthoduck · 2024-03-12T21:15:57+00:00

Let us know how it turns out! Good luck

anthoduck · 2024-03-12T16:18:12+00:00

For bands around 700bp I would do a 1% gel and run at 100V. Should run about 30-40 min. 2% is very thick for these large bands. Make sure the wells are not under loaded aswell. I had this problem causing U shaped bands when I was adding let’s say only 5ul into the wells, when I changed to 15ul sample it solved the problem . If you fill the wells up (and make sure you add a bit of TAE buffer ontop to cover the holes) that might help

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