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.

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3 thoughts on “Back to the Grind

  1. Sounds miserable. I must admit that I’d rather to nearly anything above cloning. There are so, so many places for things to go wrong. Good luck, though. I’ll send some of my lab kharma your way…since I’m taking a week off, not because I have any to spare. 😉

  2. With XbaI, one thing to always keep in mind is dam methylation.

    And holy fuck! I just looked at the NEB Web site to remind myself whether it was dam or dcm methylation that could happen to an XbaI site, and I find out that they have reformulated *all* of the restriction enzymes to work with a single buffer!

  3. I checked, this XbaI site is not subject to dam methylation.

    Also, I didn’t know about the NEB buffer change either!

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