When I came into the lab and saw that there were no colonies on any of the plates, it was a bit frustrating. No colonies meant the bacteria never took up the plasmid, which meant all the cloning work from before hadn’t paid off yet.
I wasn’t able to find pictures of the failed plates, so the picture on the right is actually a plate from a negative control (so it’s supposed to have nothing on it). But it accurately represents what the plates looked like when we walked in that morning: completely blank.
The first response was to redo the ligation. Ligation is the step where the insert DNA and plasmid backbone are glued together using a ligase enzyme, and if this step fails, nothing downstream will work.
During bacterial transformation, different amounts of ligation product were added (10 microliters and 5 microliters) to see if changing the amount helped. The bacteria were placed in the shaker for two hours this time instead of one, giving them more time to recover and take up the DNA. After that, everything was plated again and left overnight in the incubator, with a lot of hope riding on those plates.
The next day, there were finally colonies. That alone felt like a small win. Colonies were picked and used for colony PCR, which is a way to check if the bacteria actually have the correct DNA inside them. A tiny amount of each colony was used for PCR, and the rest was placed into liquid media to grow overnight in the shaker as a primary culture. Colony PCR is fast and useful because it lets us screen many colonies before spending time growing and expanding the wrong ones.
When the gel from the colony PCR was run, though, it failed. There were no bands. Not even in the positive control. That was a big clue that the problem probably wasn’t the colonies, but the PCR itself. If a positive control doesn’t work, it usually means something went wrong in the PCR setup, like the primers, the reaction mix, or the conditions. Instead of using M13 primers again, FLT3 primers were used this time to be more specific to the insert we were trying to detect.
The colony PCR was run again with sixteen reactions total. There were four colonies from the old ligation using 5 microliters, four from the new ligation using 5 microliters, four from the new ligation using 10 microliters, and four positive controls. This setup helped compare what worked best while also making sure the PCR itself was actually functioning.
This week had a lot of setbacks. Next week will be last week in the lab. Last chance to get results so I can present at the Moving Targets Symposium. Pressure makes diamonds… or it can burst pipes.
