Why do I get only white colonies that do not grow in the midst liquid? - Troubes with cloning in pGEM-T (May/20/2008 )

Hello everyone;

I'm having trouble with the cloning of a fragment of approximately 700 bp in pGEM-T. After the ligación and transformation into JM109 get mostly white colonies (3 or 5 blue). He chopped several white colonies to propagate in liquid LB + ampi (minicultive) but not grow.

Not that's what happens, I hope there is someone who can help me.

Allan. huh:

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Should not be a problem: Make sure ampicillin is not too much-- 50ug/ml final conc of amp or carb should be fine.

..

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I used a final concentration of 100 ug/ml of amp and 40 ug/ml of X-gal, as recommended by Promega for JM109.

I do not know if it is too ampicillin.

What do you think?

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Hi,

Amp concentration should be ok. Never had any problems with 100µg/ml.
But maybe it's worth a try to decrease it to 50µg/ml - who knows?

However, it might be worth checking if your protein of interest is expressed in your bacteria; it might well be that the expression within the bacteria is causing the problem - we faced a similar situation not so long ago... .


Good luck,
Cheers

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Hi all

Is it true that an excess of insert may affect the reaction of transformation?

I upload a sample of my ligación in an agarose gel to corroborate whether there was a staircase of ligación and I realised that the insert exceeded the index 3:1 that is recommended for ligation.

Could this be the cause of inefficient transfomation?

Please help me, I am desperate.

Allan

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Its hard to say, you could try different concentration, 3:1, 1:1 and 1:3 and see which is the best. For your first question, did you do any controls? Maybe your LB mixture is wrong and the condition is not suitable for growing bacteria. Try also not putting any amp as your control. This should give you some idea on what is the problem.

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No it cannot change the transformation efficiency but might reduce cloning effiency but you should still have Plasmid from your white colonies at least

Try a PCR with M13 universal primers directly on the colony and check whether the plasmid and the insert are there or not

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