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.

1 thought on “Cloning is sucking my will to live

  1. phosphatases make blunt cloning easier.

    NEB makes an antarctic phosphatase that specifically dephosphorylates the right (I can’t remember which one, it’s late) strand to make it easier.

    Additionally, have you put the PCR fragment in a holding vector, depending on what enzyme you are using for the rxn, it will put an extra a/t on the end of the fragment.

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