Ligation problem: clones survive selection but nothing in there?
Posted 04 January 2012 - 06:33 AM
How could you test your plates and medium?
Posted 05 January 2012 - 01:38 AM
Now I will measure the temperature of our thermoblock we use for the heatshock.....
Posted 05 January 2012 - 05:32 AM
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.
Posted 05 January 2012 - 05:49 AM
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 )
Posted 10 January 2012 - 04:11 AM
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?
Posted 10 January 2012 - 06:11 AM
Posted 10 January 2012 - 07:53 AM
- 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.
Posted 10 January 2012 - 07:25 PM
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.
Posted 11 January 2012 - 02:53 AM
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!