Spright

I cloned a gene into a vector. Stop codon is there. The terminator comes immediately thereafter, but unfortunately not in frame.

What will happen?

The manuals for some commercial vectors, such as pET, say that the terminator isn't quite necessary. My vector is self-made and the promoter and terminator are specific.

1. If the number of colonies after a ligation matches the number on the negative control (no insert), is there still a chance that some of the colonies are actually good?

2. I have a mixture of two PCR products that I'd like to ligate in tandem, one connected to each other, into a vector. However, one of them is capable of ligating into the vector alone. Is this ligation even worth trying? Will there be 100% preference for the small single insert, or is there rather a good chance that I obtain what I wish?

Dear all,

I ligated a 1,400-bp insert into a 3,300-bp vector and transformed this into DH5alpha cells. I obtained colonies, did a colony PCR with the primers originally used to obtain the insert, and I'm getting nothing. This isn't surprising to me because obtaining the insert to begin with had been a big challenge.

My second option, I believe, is to use vector-specific primers for my colony PCR.

Are there other options? For example, I can grow all the colonies, miniprep them, run the extract on a gel and recognise positive according to size. Would that work? Isn't it actually the safest method anyway, since it avoids false positives? Colony PCR, as we know, also amplifies ligation DNA that wasn't absorbed by the cells during transformation, right?

Could I actually skip the miniprep? Could I scrape colonies, boil them for 10 minutes in order to lyse the bacteria and then run the lysate on a gel? I would get, of course, a huge genomic DNA smear, but my 4,700-bp vector would be visible, wouldn't it?

Thanks!

"Unlike most commonly studied eukaryotes, Chlamydomonas reinhardtii is haploid, suggesting the efficacy of natural selection in this organism may be high".

(Taken from Jang and Emmerich, PLoS ONE, 2012).

Who would like to explain this declaration to me as if I were a (very smart) eight-year-old?