And as one the lab cried from the very depths of their souls: OH NOES!!!!!!1!!!!!!11!!!!!!

A new edict has come down from On High (that is, the Advisor).  From now on, complete sequences of every plasmid that is in our papers will be submitted as supplemental material.  Which means we need to have complete, annotated sequence files for every plasmid.

Advisor told me this little tidbit of news when I went in to see him with a few sample paragraphs of my Materials and Methods section which describes plasmid and strain construction.  Advisor had previously told me that he likes a very detailed M and M section.  So, I wanted to know if I had achieved the right amount of detail.  He told me that what I had would be fine because of this new rule had had just devised.  He further went on to tell me that this should not be much of an issue for me since he knows I have been making these sequence files all along, so I’ll just gather them up and if I email them to him he will convert them into genbank format.  He was not being sarcastic, the man honestly believes that I have annotated sequence files for every plasmid I have ever created (there are 62 of them, not that all of them will be in the paper only maybe a quarter of them).  I do have sequence files for most if not all.  But they are not annotated.  And if they are it’s in an obscure shorthand only understandable by me.

I casually mentioned this to a couple people in the lab and there were immediate cries of dismay.  And then I politely suggested to Advisor that he send an email detailing this exceptionally brilliant new planof his so people could start planning ahead.  Also, that I wasn’t going to spread the news because I didn’t want people to shoot the messenger.

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Back to the Grind

I didn’t really mean to take the last 5 days off from lab, it just happened.  I blame  it entirely on the fact that my husband was here and I can’t be expected to care about yeast when my husband, who is usually far, far away, is sitting in my apt.  Right?  But now he’s gone back to California, so it’s back to lab for me.

It felt really good to take a break.  Really, really good.   So good, I think I’m actually up for working and maybe even *gasp* writing.  We shall see what the day brings.

———–

Cloning:

The vector I’m trying to cut my insert out of is the product of 6 subclonings.  Let me tell ya’ it was a joy trying to figure out that mess.  Anyhoo, this insert has previously been cut out from this vector (vector 1) to make a different vector (vector 2), using the exact same method I’m trying to use.  (Before you ask, I cannot just cut it out of the 2nd vector using the same method because it was cut out using XhoI, Blunted, then cut with XbaI and then ligated into Ecl136II and XbaI.  However, I just realized, I  may be able to cut it out with EcoRI and XbaI….hmm….).  So, I thought, easy peasy, I  just do the same thing as has been done before and presto changeo, I have my vector (vector 3).

Wrong.

First, the size of the insert and the size of the vector backbone from which I was trying to liberate it were very close in size.  So, I ran a long gel and cut out the top band, because according to my calculations, the insert was bigger than the vector.  Then I did the ligation and transformation and miniprepped them and I did a digest to identify clones with insert.  But, the digest came out sort of funky.  So, I sent some clones to have the edges of the insert sequenced and lo and behold, I had ligated two vectors together.

So, I  already know there is something slightly goofy because the sizes aren’t quite right.  I recut out my insert and this time, just to be certain, I ligated both bands into my new vector (you know, in separate reactions).  Then, I did a transformation and I miniprepped them and did a digest and goddamnit, neither possibility looked right.  It looked like the enzymes I was using to check for positive clones weren’t cutting at all, even though they linearized the parent vector just fine (which is what I expected).

So, fuck it.   I’m PCR amplifying the damn thing from a different vector (vector number two in the first paragraph; a vector I’ve actually used in an experiment so I know it’s good) with sites on the ends.  No more Ms. Nice Girl.

Except, I’m waiting on primers.  I ordered the damn things before Christmas, when will they get here?

But, I’m intrigued by my new idea of cutting the insert out of vector 2 with EcoRI and XbaI.  Maybe I’ll get started on that today.

Dedication or Stupidity?

It is the week of Christmas.

The weather has turned seven shades of ridiculous.

The boss is out of town for the week.

My entire lab is here working (except for, you know, the boss).

And, just on a personal level, my husband is in town and I still don’t have all of my Christmas shopping done.

What the hell are we doing here?

———-

Thesis:

So, my advisor already made CPP’s suggestion of doing all of the figures and figure legends first.  However, after spending hours of time moving images miniscule distances to the right or left and changing the font on the figures several times, I had to accept that I was procrastinating.  So, I thought I should start writing the text of the Results.  I still have a couple of figure legends I could write, though.  Maybe I should do that.  Especially since all I’ve really done is change forms of procrastination.

I’m not even thinking about the introduction.  There is no introduction in my fantasy world.  Panic does not even come close to describing how I feel about the introduction.  I’m just going to get through the results first.

———

Sleep:

I’ve got the sleeping pills.  They helped me get to sleep, but I didn’t stay asleep for the first couple of nights I took it.   Then, I started sleeping through the night and needing to sleep through half of the day, too.

So:

No sleeping pills = no sleep, tired all the time

+ sleeping pills = lots of sleep, sleepy all the time

Not sure this is an improvement.  I think I’m going to try a different sleeping pill and see if that’s any better.

———

Finally:

If someone could make my super simple subcloning work right, I’d appreciate it.  I really don’t understand what the problem is.   Cut fragment out of vector 1, ligate into vector 2, transform, miniprep.  But, then I do a digest to check if it’s right and the digest comes out all wrong.  On all of them.  This is the second time I done this damn cloning.  If I have to do it a third time, I’m going to be wicked pissed.

Cloning is sucking my will to live

Okay. Here’s the deal. The reason I started this here blog is because I needed a place to rant. I decided this after a particularly nasty debacle with mislabeled, poorly made vectors which I alluded to here. I started this blog about two days later, so that should give you some idea of the timeline I’m working with here.

Since then, I have been trying to clone an insert into a pUC19-based vector (which has been properly constructed–I checked). The thing I’m trying to do is put in a PCR fragment with a BamHI site at the 5′ end and a blunt, phosphorylated 3′ end. For various reasons, I can’t use any of the blunt cutter sites in the MCS, so I have been cutting with SphI and blunting using T4 DNA Polymerase.

It won’t work. I have tried and tried and tried. Why it doesn’t work is a complete and utter mystery to me. I suspect that the blunting reaction isn’t working as planned. I much prefer Klenow but that blunts different overhanging ends and T4 is supposed to be the one to use in this instance. I have done this kind of thing millions of times in the past, I don’t understand why this one is so damn difficult!

My apologies to the people who have no clue what I’m talking about. I just needed to vent my frustration about this stupid thing.

This just in!

Scientist Enters Incorrect Incorrect Info on Own Plasmid, Screws Self

Mrswhatsit, a senior graduate student at WantstobeHarvard University, discovered this afternoon that her cloning project will not work because the information in the database about a particular vector was incorrect.  Sadly, this information was entered by Mrswhatsit herself.  Mrswhatsit freely admits her responsibility in this mix-up.  “It’s my own  fault,” she said, “I fucked it up.”

Mrswhatsit has been plagued with difficulties throughout this cloning project.  Up until today, these difficulties have centered around other people’s fuck-ups (one vector not only had the wrong gene, the gene was from the wrong species).  These problems have caused the cloning project to drag on and on, wasting precious time.  After spending a month trying to sort out the various misinformation surrounding the starting plasmids (including good old-fashioned sequencing of the plasmids), Mrswhatsit was looking forward to finally getting the damn cloning finished.  But it was not to be.

The cloning project is an ambitious one, involving the insertion of multiple genes, done in a sequential fashion.  While the first step of the cloning procedure seemed to go okay, the second step tanked.  “Essentially, I was getting an insert cut out when I was just trying to linearize this construct which was the product of the first cloning step.  Since I thought I knew what the sequence was, I couldn’t figure out why this was happening.  Finally, I looked over the sequence files for the insert from the first step and lo and behold, there are restriction sites I didn’t think were there.”  Mrswhatsit had tried to be clever and PCR amplify her insert from a construct she made several years ago.  “The extra sites hadn’t mattered when I first made this construct, but now that I was trying PCR amplify something from it, it mattered a great deal.  I went back to my lab notebook and found that the way I made the vector was not the way I reported it in the database entry which was what I was basing my cloning strategy on.  I have corrected the database entry, but that doesn’t give me back the weekend I spent in the lab trying to do this damn cloning.”

Mrswhatsit credits her organized lab notebook for helping her get to the root of the problem.  “If I hadn’t had a table of contents, it would’ve taken me hours to find that details of making that old construct.  As it was, it took me about 10 minutes.  Too bad I didn’t look it up earlier.”

When asked for advice for future lab generations, Mrswhatsit had this to say, “Trust no one–and that includes yourself.”