Troubleshooting Gateway Cloning

After struggling with a particularly annoying problem in the lab, I finally got my experiment to work this weekend! When I crowed about this to my partner, he told me that whenever he figures out the solution to a tricky problem in his line of work, he blogs about it, for the sake of others who might have the same issue. I did my share of Googling while troubleshooting my experiments and didn't find a solution, so I thought this was worth blogging about. The following will make very little sense if you don't do molecular cloning.

I spent almost three months trying to clone my DNA sequence of interest into a mammalian expression vector using Invitrogen's Gateway Cloning system. Things were going smoothly for a while -- I made a pENTR entry clone, chose a pDEST destination vector, did the LR clonase reaction according to the instructions in the kit, did a diagnostic restriction digest to confirm that my plasmid had the expected sequence, and then... I couldn't maxi-prep the plasmid. This was my first time using the Gateway system, so I expected to hit some bumps in the protocol, but I've been doing maxi preps (with Qiagen kits -- regular and the HiSpeed) for years. So, I felt pretty stupid when my experiment broke down at that point. I got my plasmid out of a mini prep, but failed to recover the plasmid after inoculating a maxi-sized culture with the same exact construct in the same exact competent cells. What the hell?

As I grew increasingly frustrated, my adviser had the outlandish idea to repeat the experiment with good controls. So I did. I used every control I could think of. I transformed, mini-prepped, and maxi-prepped: my entry vector, a known entry vector with a similar sequence that we had lying around the lab, my expression vector, a similar expression vector from the lab, and the empty entry vector. I also repeated the LR clonase reaction with my plasmids and with other Gateway plasmids we had on hand.

I could get maxi prep DNA from everything except LR reaction products (i.e., any sequence of interest flipped from pENTR into pDEST through attR/attL recombination). For some reason, the expression clones would grow in mini preps, and some sort of antibiotic-resistant bacteria would still grow in my maxi cultures, but I would get abysmal plasmid DNA yield from the maxi preps. I tried growing my bacteria at a lower temperature over a longer amount of time, which sometimes helps bacteria keep larger plasmids (my plasmid of interest was about 9 Kb), but that didn't help. I bought fresh new LR clonase enzyme, but that didn't help either.

Eventually, senior lab members remembered someone having a similar problem a few years ago. They thought that the issue was finally resolved by making fresh pDEST vector by culturing bacteria containing the empty vector from the original glycerol stock. I thought that made no sense, but it was easy enough to dig the glycerol stocks out of the freezer, grow up some cells, and prep the DNA. So I did. I then used the newly made pDEST DNA to repeat the LR clonase reaction with my entry clones, transformed the LR product, and grew up some bacteria for a maxi prep.

And... it worked!

I don't know why it worked. I guess somehow, after being stored in the freezer for a while and going through enough freeze-thaw cycles, the pDEST DNA that we had stockpiled went bad. It worked well enough to take up the insert from my pENTR clones and grow in competent cells in a 2 mL culture, but then it petered out when I tried to grow a 150 mL culture of the same cells. So, if you are having a similar problem, I say to you: find your old glycerol stocks. My labmates say that the last time they had this problem, transforming the plasmid back into some ccdB-tolerant cells (the pDEST vector we used contains the ccdB suicide gene) and re-prepping from them also worked, but I can't verify that.

Next up: transfecting my expression vector into some mammalian cells to see what happens! (Also known as: the experiment that I actually care about, which I was sure I would have gotten to by now...!)