DNA cloning: White colonies on plate are unable to grow out in LB media. What am I missing?

Question

I'm using TA cloning kit from invitrogen. The plasmid is pCR (if memory serves me properly).
I amplify the amplicon from bisulfite treated DNA, and cut my band out of LM agarose. I perform a gel purification, and perform an overnight ligation. I do a transformation the following day, and grow the bacteria out on an ampicillin plate. I see mostly blue colonies, and some white colonies.
I send out the plates (overnight delivery) to a Texas company: Seqwright. They pick white colonies (8-12) which I have circled for them and grow out in LB media containing ampicillin (maybe kanamycin, too, I not sure).
On one occasion Seqwright has been unable to grow out the picked colonies. All "inserts" were batched in the way I described above. All failed. Other batched inserts processed in the same way, are able to grow.
What did I do to that one batch I sent out for sequencing? Why did white colonies not grow out in media. (I triple checked all steps).
All advice is welcomed.

Answer 1

A couple of possibilities that you might not have considered are...

1. freezing damage in shipment could have made the colonies not survive.

2. The posibility that it was NOT your error and that the lab chose the wrong plate medium to grow the cultures...perhaps with the wrong antibiotic in the medium.

If your procedure was the same then the results should be the same. If not then a variable not consdered is involved. It may not be your variable that was at fault. Similar things have happend to me in processing blood tests ordered that the results were invalid due to the wrong tube used for collection or process medium for growth.

Answer 2

I don't follow you exactly, but possibly the white colonies are a contaminant, not an actual clone. When white colonies are picked and they don't grow, it's usually a sign of a contaminant and something is wrong with your amp plates. Or perhaps the company picking your colonies is using the wrong antibiotic, or you have some type of problem with the origin of replication; or for some reason your DNA is toxic to the E.coli.

Answer 3

I have had that happen to me. I had picked white colonies, yet when i had them sequenced my insert wasn't ther. One of the explainations provided in the Invitrogen manual is as follows:
"Hundreds of colonies from the vector + PCR insert reaction should be produced. 95 (+/- 4%) of these colonies will be white and 90% (or more) of these will contain the 750 bp insert when analyzed by EcoR I digestion and agarose gel electrophoresis.
Relatively few colonies will be produced in the vector-only reaction and most of these will be dark blue. You may observe a few white colonies. This results from removal of the 3´ deoxythymidine overhangs creating a blunt-end vector. Ligation
(re-joining) of the blunt ends will result in disruption of the LacZα reading frame leading to the production of white colonies."

Here is the link for the manual PDF
http://www.invitrogen.com/content/sfs/manuals/topota_man.pdf