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Ligation problem: clones survive selection but nothing in there?


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24 replies to this topic

#16 phage434

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Posted 04 January 2012 - 06:33 AM

And added to the medium AFTER it is cooled...
How could you test your plates and medium?

#17 Moppelkotze

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Posted 04 January 2012 - 06:36 AM

Oh yes, of course, after it's cooled.

#18 Moppelkotze

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Posted 05 January 2012 - 01:38 AM

So, I tested again our plates, water, LB broth, DNA: no contamination.

Now I will measure the temperature of our thermoblock we use for the heatshock.....

#19 phage434

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Posted 05 January 2012 - 05:32 AM

Where are your competent cells coming from? In my experience advising people, this is the problem in 75% of the "ligation is failing" problems.
Test their competence. Transform 100 pg of pUC19, and you should get hundreds of colonies. It sounds as if you have done this, and it doesn't. This is telling you directly that the cells are not working.

#20 Moppelkotze

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Posted 05 January 2012 - 05:49 AM

I really hope, that the OneShot Top10 E. coli from Invitrogen are worth the money (they are pretty expensive, but hey, it's not my money... I once suggested to make competent DH5alpha, but my boss doesn't want to take a risk, never change a running system, and so on).

Yes, you're right, we tested them with pUC19 and had only few colonies. But now: Where should I search for the problem?

Both Thermoblocks we use produce 40 °C when set to 42 °C. I need another thermometer to check this. But it's unlikely that both thermomixers stopped working properly at the same time, isn't it? They are from eppendorf (I don't want to show off here, just want to tell you that quality of the equipment is presumably not a problem ;) )

#21 Moppelkotze

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Posted 10 January 2012 - 04:11 AM

Update: I had about 5 colonies, on my pUC19 plate, where I expected to have 100. We set our thermoblock to 44 °C and measured 41,5 - 42 °C with a liquid thermometer.
My colleague tried aswell a transformation with kanamycin resistance and had a few colonies (during the last weeks she never got any colony on kanamycin).

We will analyze the colonies tomorrow.

What else can I change to obtain a good efficiency again? Is ist worth to measure the pH of the LB broth and also of the the agar?

#22 phage434

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Posted 10 January 2012 - 06:11 AM

So, are you using the Invitrogen One-Shot cells? These come with both a pUC19 control and with SOC for resuspension. Are you allowing sufficient time for antibiotic expression prior to putting them on selective plates? Why don't you tell us exactly what you do in your transformation (and control).

#23 Moppelkotze

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Posted 10 January 2012 - 07:53 AM

Ok, of course I will tell you the protocol. For the positive pUC19-control I used everything from Invotrogen, the plasmid and the SOC.

- thaw the cells on ice
- put the cells to the DNA (I know I shouldn't pipette the Top10 cells, but I made no bad expirience with it. I use a 1000 µl tip to avoid big shear stress)
for pUC19 10 pg
- 30 min incubation on ice
- 30 s (for pUC19) 40 s (kanamycin vector) heatshock on a thermomixer (I hope with 42 degrees)
- 2 min on ice
- add 250 µl prewarmed SOC (for control and kanamycin-trafo)
- 1 h 37 °C shaking at 180 rpm (usually we use 200 rpm, but this wasn't possible yesterday) Eppi lying on its side
- plating on agar with the appropriate AB (1:10 dilution with LB and spread 30 µl + 500 µl)
- over night incubation at 37 °C (inverted)
- 6 colonies on the 30 µl plate, 120 colonies on the 500 µl plate

Hope this is enough detail.

#24 phage434

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Posted 10 January 2012 - 07:25 PM

So, your efficiency is rnot all that bad. You start with 10 pg of pUC19 DNA. This is 1e-5 ug. Your final volume, if I follow you, is 3 ml (50 ul cells, 250 ul SOC, diluted 10:1). You plated 30 ul, or 1e-2 of the reaction, and got 6 colonies. This is 6e7 cfu/ug. Your 500 ul plate showed 500/3000 = 1/6 of the final volume, with 120 cfu. So this is 720e5 or 7.2e7 cfu/ug, a consistent number. I would hope this would be more like 5e8 cfu/ug, but it is only off by 10x or so.

You probably can improve this a good deal by switching from 1.6 ml eppendorfs for transformation to a 2 ml eppendorf. These allow much better aeration during the recovery. Make sure your DNA and cells are well mixed. I'd recommend tapping the tube and then spinnng it down.

I don't see why you are diluting with LB prior to plating. You need as many cfu on a plate as you can get, and plating the undiluted reaction will work.

#25 Moppelkotze

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Posted 11 January 2012 - 02:53 AM

The transformation efficiency is better than before, that's right. But 10x reduced efficiency is quite a lot, when I didn't change anything in my protocol. On the one hand I want to fix the problem, so I will give the 2 ml eppendorf tube a try. But on the other hand I also want to find out what causes the problem. I have to generate a transgenic mouse, there's a lot of cloning work waiting for me.

Oh sorry, this was my fault: The dilution step was only performed for the pUC19-control, for the real transformation experiment we don't dilute anymore. (in the past we plated 10 µl, 100 µl and the rest of the transformation, now we plate a small part (e.g. 30 µl) and the rest)

Thank you a lot for your help so far!




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