can't be out-matched, can't be out-run
Mar. 5th, 2011 08:53 pm![[personal profile]](https://www.dreamwidth.org/img/silk/identity/user.png){kind=small}
asimplechordHangtimes at ![[livejournal.com profile]](https://www.dreamwidth.org/img/external/lj-userinfo.gif)
eckerlilas' today! For all that we all live in/around H-town, we don't get together enough when we're not standing in line to get into a show. We def should pick a weekend and an actor and have a movie marathon soon.
Also? I cleaned out my closet and cleaned off my bookshelves today. Tomorrow we tackle the kitchen cabinets, and then we'll take things to Goodwill, to the public library, and to be recycled.
Random bullet-points about the Linkin Park show the other night:
In which I ramble about work (again). Well, mostly about Student.
He's having cloning issues. Not even, you know, DIFFICULT cloning issues. All he had to do was take oligos for shRNA (which he didn't even have to DESIGN, someone else had already done so and used them in a different vector background than the one he wanted), anneal them, ligate into the cut vector, transform some DH5a, miniprep some plasmids and verify via diagnostic digest that they had the insert, and sequence to verify that he really did use the right oligos to insert.
He was fine through the digest (although I think he lost brain cells while away on vacation, because he needed me to talk him through each step, including a reminder that he needed to add buffer to the plasmid + restriction enzyme during the digest), but when he sent two of his samples for sequencing, he got ~400 bp of good sequence that then dropped to no signal right where his insert should start. I explained that that was a secondary structure issue, and he could call the sequencing facility and ask them to add extra DMSO or whatever they normally do when there's a hairpin or other structure that polymerases have a hard time reading through.
He did that, and got the same result.
Now, the way shRNA *works* is via hairpins, creating secondary structure that RNA polymerase can't read through, and thus stopping protein synthesis by preventing the RNA from ever being processed. But I have not had secondary structure so strong that I could not get sequence from the parent plasmid before.
In any case, he kept going on & on about how he didn't understand why he wasn't getting good sequence when the diagnostic digest was fine. I could not get him to understand that these were two ENTIRELY DIFFERENT methods, and one did not necessarily correlate with the other.
I just. BASIC MOLECULAR BIOLOGY.
HE SHOULD KNOW THIS BY NOW. HE'S NOT A FRESHMAN TAKING BIO101.
I suggested Student talk to our boss, because he has far more experience than I do with recalcitrant cloning and sequencing.
He didn't want to do that. So I suggested he talk to Post-doc W, who designed these oligos and used them in another vector background.
Well. It turns out that instead of taking the individual oligos and annealing them, then using immediately (as the protocol's directions suggest), he decided to use her month-old annealed and diluted oligos. Because he didn't understand that thawing them without going through the heating and re-annealing could lead to secondary undesirable secondary structures, and since they are two oligos *designed* to make hairpins, they can anneal on themselves in more than one way, and the inappropriate structure would not be detectable on a diagnostic gel because the primers are small enough that two or three of them won't look different from the insertion of one of them, as long as the ligation happens and creates the diagnostic restriction site.
I repeat: BASIC MOLECULAR BIOLOGY. He is a grad student. He should know this by now.
IDK, I feel like he is not PhD material at this point, and he needs to shit or get off the pot, but I am not the person who has the authority to give him a come-to-Jesus talk.
OK. I am going to go drink some Left Hand Milk Stout, be jealous that J went to NYC and saw American Idiot (again), and read an emailwhy_me_why_not sent me earlier.
eckerlilas' today! For all that we all live in/around H-town, we don't get together enough when we're not standing in line to get into a show. We def should pick a weekend and an actor and have a movie marathon soon.Also? I cleaned out my closet and cleaned off my bookshelves today. Tomorrow we tackle the kitchen cabinets, and then we'll take things to Goodwill, to the public library, and to be recycled.
Random bullet-points about the Linkin Park show the other night:
- Someday I will get A down into the pit again. He had a bad experience at a Less Than Jake show (it's all fun and games until a crowd surfer gets dropped on your head and you trip on the banana the lead singer tossed out into the crowd earlier) and hasn't been too keen on being up close and personal. Most of the time I don't care, and if it's a show where I'm aiming for the barrier, he generally doesn't come with, but at least for this show, even if he only wanted to hang out back by the sound booth, at least he didn't insist on an actual seat up in the bowl.
- Is Linkin Park getting more obviously political? I don't remember their early albums being overtly so, but Minutes To Midnight was, and the media they put on A Thousand Suns definitely is.
- Loved the screens, including the display of Mario Savio's put yourself upon the gears and the wheels speech to start the show, and MLK Jr's wisdom, justice, and love bit for the encore, plus the Oppenheimer "I am become death".
- I think this album works at an indoor venue. I'd always seen them outside before, at amphitheaters and stadia where the power and pyrotechnics of their previous albums showed to their best advantage, but this was a good show. In terms of sound quality, it was probably the best show I've seen at Toyota Center.
- I'm curious to see if the Flash Seats thing continues. Useful in that tickets aren't easily lost if you don't HAVE them, but I'm not that keen on having a set ID and/or credit card required to enter the venue.
- Is Mike Shinoda aiming for Justin Long or Justin Bieber with that haircut?
- NRGH, Mario Savio's speech into Papercut into Lying.
- Good job splicing songs from albums together.
- The harmony on The Catalyst was gorgeous live.
- LOL, Phoenix in a cowboy hat. Did he think they were heading to the rodeo when the last show of the tour was done?
- Not so much, Chester Bennington's voice on Shadow of the Day. Also, do they owe U2 royalties for ripping off Still Haven't Found What I'm Looking For there?
- Paper Tongues as openers. Much better than when they supported Bush back in October. Technically proficient. I am not that fond of the single they've played on the radio here, but if they put out an album that was more like Ride To California, I'd buy it.
In which I ramble about work (again). Well, mostly about Student.
He's having cloning issues. Not even, you know, DIFFICULT cloning issues. All he had to do was take oligos for shRNA (which he didn't even have to DESIGN, someone else had already done so and used them in a different vector background than the one he wanted), anneal them, ligate into the cut vector, transform some DH5a, miniprep some plasmids and verify via diagnostic digest that they had the insert, and sequence to verify that he really did use the right oligos to insert.
He was fine through the digest (although I think he lost brain cells while away on vacation, because he needed me to talk him through each step, including a reminder that he needed to add buffer to the plasmid + restriction enzyme during the digest), but when he sent two of his samples for sequencing, he got ~400 bp of good sequence that then dropped to no signal right where his insert should start. I explained that that was a secondary structure issue, and he could call the sequencing facility and ask them to add extra DMSO or whatever they normally do when there's a hairpin or other structure that polymerases have a hard time reading through.
He did that, and got the same result.
Now, the way shRNA *works* is via hairpins, creating secondary structure that RNA polymerase can't read through, and thus stopping protein synthesis by preventing the RNA from ever being processed. But I have not had secondary structure so strong that I could not get sequence from the parent plasmid before.
In any case, he kept going on & on about how he didn't understand why he wasn't getting good sequence when the diagnostic digest was fine. I could not get him to understand that these were two ENTIRELY DIFFERENT methods, and one did not necessarily correlate with the other.
I just. BASIC MOLECULAR BIOLOGY.
HE SHOULD KNOW THIS BY NOW. HE'S NOT A FRESHMAN TAKING BIO101.
I suggested Student talk to our boss, because he has far more experience than I do with recalcitrant cloning and sequencing.
He didn't want to do that. So I suggested he talk to Post-doc W, who designed these oligos and used them in another vector background.
Well. It turns out that instead of taking the individual oligos and annealing them, then using immediately (as the protocol's directions suggest), he decided to use her month-old annealed and diluted oligos. Because he didn't understand that thawing them without going through the heating and re-annealing could lead to secondary undesirable secondary structures, and since they are two oligos *designed* to make hairpins, they can anneal on themselves in more than one way, and the inappropriate structure would not be detectable on a diagnostic gel because the primers are small enough that two or three of them won't look different from the insertion of one of them, as long as the ligation happens and creates the diagnostic restriction site.
I repeat: BASIC MOLECULAR BIOLOGY. He is a grad student. He should know this by now.
IDK, I feel like he is not PhD material at this point, and he needs to shit or get off the pot, but I am not the person who has the authority to give him a come-to-Jesus talk.
OK. I am going to go drink some Left Hand Milk Stout, be jealous that J went to NYC and saw American Idiot (again), and read an email
why_me_why_not sent me earlier.
