I have been a resident of what graduate students refer to as "cloning hell" for over 2 weeks now. It seems simple enough. I need to take two pieces of DNA, cut them and then paste them together to make one big piece. Why can it never be as simple as it should be??
First I had background issues. My uncut control DNA (negative control) turned out to be just as good at getting into bacteria as my cut DNA. A few extra gels and digests and I got that under control.
Now I don't get ANY bacteria colonies! What's up now? Another gel later I discover that my insert DNA has disappeared. It was there after the PCR amplification and after the digest, but somewhere between getting cut and getting pasted it disappeared. So now I'm re-doing the PCR amplication step - basically back to square one. Argh.
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