Troubleshooting 101
It looks like I’ve hit my metaphorical stumbling block.
The miscreant samples that I previously mentioned– the ones that didn’t work out perfectly in the first try– have been giving me more headaches than I care to blog about. But here’s a (not so brief) summary; please excuse me if I start ranting…
After the first round of sequences were analyzed, I tried re-PCRing the ones that didn’t amplify correctly. Some worked the second time, but most of the samples still showed nothing. So I took a step back, and I re-extracted those samples and then ran PCR on the new extractions. But nope, no luck there either. So I ran a whole-genome amplification on these samples, hoping to increase my DNA concentration and bypass the issue of bad tissue samples. But STILL no luck; after running PCR on the whole-genome samples, it appears that while I did have success in increasing my DNA concentration, the troublemaking samples still won’t amplify.
I’ve run out of bullet points on my list of things to do if the samples don’t work…so I’m racking my brain to think of what has been going wrong. Right now I’m trying PCR again, but with a lower annealing temperature…or maybe my reagents are weird and I need a new stock. Hopefully I’ll think of something else to test or find out what’s actually going wrong with my rogue samples.
Either way, I’m feeling the pressure of deadlines (abstracts are due next week, and posters next weekend) and the stress of many failed attempts. And now I’m wondering…what happened to my summer vacation?