3 replies to this topic

[Dino]

Hi All,

I've been trying to clone a gene into the pGEMTeasy vector and was successful in getting colonies and isolating plasmid.
When I perform PCR using my target gene primers I also get a nice clean band.

When I initially plasmid prep'd the samples using a low copy vector protocol (to get a higher yield)
the negative control (a background colony, blue) always ends up with a significantly higher concentration
around 150-200ng/uL however the clone always purifies at 60-80ng/uL.

I've tried this on two different occassions, each time purifying two clones and one background colony.

Any ideas as to why I'm getting low yields for the clone and not for the background colonies?
I'm using DH5alpha home made and the vector is commercial.

Could the insert going in backwards have any effect on plasmid copy number?

Thanks!
D

[bob1]

The insert shouldn't have much effect on the copy number unless it is producing a product that is toxic.  Assuming that there aren't any start codons if the insert is backwards, would mean that there shouldn't be any effect at all.

Size of the plasmid can have a role on the amount of DNA produced, but I don't know the mechanism.

[Dino]

Hi Bob,

Yes I also thought that perhaps the product is toxic. In any case I'm getting a PCR product at the right size and my digestions are showing a band at the right size, no other non specific stuff going on. Sending for sequencing this week. Its a fairly routine insert, ~1100 b.p. so I doubt it would affect the yield. I was thinking perhaps DH5alpha may not be fully compatible with pGEMT easy? Anyways fingers crossed this is the insert I need.
Thanks Bob.

[Dino]

Just to update. I switched to old school plasmid preps. Using isopropanol and 70% ethanol to clean my plasmids. Avg of ~1ug of DNA 260/280 ~1.7-1.9

I think our kit is probably off, pH or something. Glad I worked it out! Thanks for the suggestions.

D