Can I use bacteria to make a cDNA library containing eukaryotic plasmids?

Question

standard competent cells

Answer 1

Sure. You say that the cells are competent.

Just clone your cDNAs into your plasmids, and then perform transformation (chemical or electrical, your choice).

Electrical transformation's more fun, though I have caught a specimen on fire that way.

Bacteria aren't picky about which plasmids they intake, just make sure that the plasmid contains a bacterial replication promoter so that the bacteria knows to duplicate it while performing mitosis.

Also, plasmids in excess of 12kb length have trouble getting into bacteria, so be warned.

Hope this helps.

Answer 2

Usually eukaryotic plasmids are designed so that they can also be propagated in E.coli, so that you can easily get large amounts of purified constructs.

So provided that you have an appropriate antibiotic restistance gene and a bacterial origin of replication yes.

If however it is some special vector that doesn't have these two features, then NO you cannot use them-even if transformation is successful the plasmids will fail to replicate and thus they will be diluted out of the bacterial population.

Answer 3

Absolutely.

Answer 4

c) I think. Normally they place the bacteria in a culture with ampicillin and if the bacteria contains the recombinant plasmid it will be destroyed as the gene of interest will have disrupted resistance to ampicillin.

Answer 5

yes.

Answer 6

yes...