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[Cdmc]

Hey everyone,

I have been having problems with transforming and eventually preparing maxipreps of pHellsgate 12 from DB3.1 cells. Oddly, this does not happen when I deal with other Gateway cloning plasmids like pStaling, pStargate, pWatergate and pOpOff vectors.

The problem is the slow growth on the LB plates after transformation. This is our lab protocol:

1. Incubate competent DB3.1 cells with the plasmid for 10 minutes on ice.
2. Heat shock at 42C for 3 minutes.
3. Incubate at room temperature for 10 minutes.
4. Add 1 mL of LB on the cells and incubate at 37C with shaking for 1.5 hours.
5. Plate onto LB spectinomycin plates. (LB contains 0.5% yeast extract, 1% NaCl and 1% bacto-tryptone)
6. Incubate at 37C.

Colonies would usually apear after 2 days or more of incubation, which is longer than what I am used to. Preparing liquid cultures on LB with spectinomycin also takes 2 days or more. This is also longer than usual (overnight for other plasmids).

After doing the alkaline lysis method for maxiprep and CsCl ultracentrifugation, I barely get enough plasmid. Sometimes, I do not get plasmid at all.

I do not know what I am doing wrong. I hope someone can help me.

Thanks!


Sincerely,
Chris