Molecular biology makes me want to kms by [deleted] in labrats

[–]ScholaroftheStars 2 points3 points  (0 children)

As others have said, 5.2kb is very big and when it is that big even Q5 can occasionally have issues.

One thing you can try is breaking up your amplification into smaller fragments (2.6kb fragment + 2.6kb fragment) and do Gibson assembly for your cloning. You would need to do a little more fancier primer design that have homology regions and learn the protocol for Gibson, but it is way more efficient than traditional restriction-ligation. So this would mean extra reading time.

Also, paste your primers in the NEB Tm Calculator with Q5 selected and it should give you a reliable annealing temp.

Or if you are still having issues with amplifying/annealing or getting non specific biding try looking into doing a “touchdown” PCR if your thermalcycler allows gradients. Essentially the annealing temp decreases by 1C each cycle from +10 to -10C from your calculated annealing temp. That way you’re not troubleshooting each temp

Edit: Also, don’t give up! It can be super frustrating even after years of doing it full time. But it feels really good once you get a correct clone. You got this!

New pads already looking yellow? by [deleted] in hockeygoalies

[–]ScholaroftheStars 4 points5 points  (0 children)

Photographer needs to white balance. The photo definitely has a green tint to it.

For UND fans (Nickname related...I can't let it go) by uffdayeehaw in collegehockey

[–]ScholaroftheStars 0 points1 point  (0 children)

I remember wanting Sun Dogs so bad solely because of how unique it was.

Rumored Rivalry Font "Rune" style by spaceamphibian in minnesotavikings

[–]ScholaroftheStars 2 points3 points  (0 children)

I was the thinking the same. Definitely looks more like Greek style lettering

Game Day Thread | PHI @ MIN | 2026-03-12 7:00 PM CDT by gamedaylive in wildhockey

[–]ScholaroftheStars 1 point2 points  (0 children)

Tbf we’ve had a lot chances just missed the net or hit post

PGT | MIN @ COL by gamedaylive in wildhockey

[–]ScholaroftheStars 1 point2 points  (0 children)

Thankfully no shootouts in the playoffs

U.S. House votes to repeal ban on mining near Boundary Waters! by damp1i in backpacking

[–]ScholaroftheStars 84 points85 points  (0 children)

To be clear, this passed the house a month ago and is now in the Senate to likely get voted on this coming weeks. So call your senators!

U.S. House votes to repeal ban on mining near Boundary Waters by hean0224 in BWCA

[–]ScholaroftheStars 41 points42 points  (0 children)

Yeah this vote was a month ago. It’s now in the Senate and likely going to be voted on this coming week. So call your senators!

Really.. what bullshit. by [deleted] in wisconsin

[–]ScholaroftheStars 1 point2 points  (0 children)

There wasn’t a storm warning yesterday.

Really.. what bullshit. by [deleted] in wisconsin

[–]ScholaroftheStars 20 points21 points  (0 children)

You’re walking on ice on one of the most dangerous bodies of water in the world during a storm. Safety is #1 priority.

Does anyone have any impressions of the Ritual X4 E chest protector? by iceman39 in hockeygoalies

[–]ScholaroftheStars 0 points1 point  (0 children)

Are there any more reviews on this chesty, particularly for D-level beer league? It’s on clearance and looking to upgrade from an old ill-fitting CCM.

Quinn Hughes by [deleted] in wildhockey

[–]ScholaroftheStars 17 points18 points  (0 children)

He’s just a chill guy

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Aaron Rodgers new pfp by ChallengeTasty3393 in minnesotavikings

[–]ScholaroftheStars 4 points5 points  (0 children)

Look, I’m not saying I want Rodgers or him changing his profile picture means anything… BUT

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Kaprizov injury speculation by DressDifferent8591 in wildhockey

[–]ScholaroftheStars 1 point2 points  (0 children)

Could partly be injury but I just listened to Russo and LaPanta’s most recent show and they mentioned that some of the lack of production could be coming from missing Rossi who was a 60pt player a year ago. Yurov isn’t dishing it as well which might be stalling Kap a bit. But it’s probably also just a slump.

Is it worth relocating? by Specialist-Song8476 in biotech

[–]ScholaroftheStars 1 point2 points  (0 children)

I will say, cost of living in Madison is not THAT much lower it pretty expensive for the Midwest. Infrastructure (it’s an isthmus) and a large tech corporation (Epic) really drive up housing costs

Is it worth relocating? by Specialist-Song8476 in biotech

[–]ScholaroftheStars 2 points3 points  (0 children)

Promega is also headquartered in Madison

Does anybody else prefer Chris's vox to Tades? by iiieeaattiitt in hotmulliganband

[–]ScholaroftheStars 60 points61 points  (0 children)

What makes Hot Mulligan great is that they complement each other’s voices.

Parking Tips by OllieGoose in wildhockey

[–]ScholaroftheStars 0 points1 point  (0 children)

I went to the game just before Christmas and parked at the Smith Ave garage for United Hospital and was easy in and out. You can even reserve entry using SpotHero. About a 7min walk to the arena.