Transformation problem. Please help! - 1.7kb insert to 5.4kb vector; double digested w/ EcoRV & XhoI (Aug/04/2009 )

Hi all.
I've been struggling with one transformation for months and I really need some advices from you guys.
This is my case:
I have an 1.7kb insert and it has been double digested w/ EcoRV and XhoI to create a blunt end and a sticky end. I performed the same (double) digestion on the vector (pcDNA3.0-FLAG, 5.4kb).
Next, I performed the ligation (so far I'd tried 2 ligation kits: Takara DNA Ligation Kit & Fermentas Rapid DNA Ligation Kit) and then I immediately made the transformation into DH5a cells.
However, I can rarely get colonies w/o insert using Takara Ligation kit and no colonies at all using Fermentas DNA Ligation kit. I have no idea what had gone wrong since I followed the protocol accordingly.
I'll write down the steps which I'd done during the ligation and transformation. Please comment.

Ligation - Takara kit
Different ratio of insert:vector volumes (from 10:1 to 3:1) had been tried.
Same volume of ligation buffer was added and mixed

The mixture was incubated @ 16degC for 30min
10ul of ligation mixture was used for transformation

Ligation - Fermentas Kit
3:1 of Insert:vector (volume ratio) had been tried so far
4ul of 5x ligation buffer was added
1ul of T4 DNA ligase was added
Autoclaved H2O was added to fill the ligation mixture up to 20ul

The mixture was mixed and incubated @ 22degC for 5min
5ul of ligation mixture was used for transformation

Transformation (DH5a)
Each tube contained 50ul of competent cells was used.
The cells were thawed on ice for 10min after taking out from -80degC
The ligation mixture was added directly into the tube
The tube was rested on ice for 30min
The cells were heatshocked @ 42degC for 20sec
The tube was rested on ice for 2min
950ul of prewarmed LB was added to the tube thereafter
The cells were incubated @ 37degC waterbath, shaking @ 300rpm for 1h
200ul of the mixture was spreaded to LB plate containing ampicillin (twice)

TIA.

Ligation of Blunt end DNA is fa less efficient than sticky ends Promega use to have a great ligation buffer which contains PEG and was in my hand working very well with Blunt ends. Be sure to lower the temperature of the ligation due to the movement of molecules are higher at temperature above 4°C. Maybe I would try a ligation overnight at 4°C with a buffer containing PEG.

I always perform blunt end ligation at 16°C over-night whatever ligase I use.

little mouse on Aug 4 2009, 09:01 AM said:

I agree - I always do a 16degC ligation overnight for blunt ends. You can also add 2ul of 50% PEG 4000 (for a 20ul ligation) to increase the chances of the blunt ends ligating (it worked for me in my magic 3 way ligation with blunt ends!).
Good luck
Clare

I also do a ligation at 16°C overnigt and the result is very good. In transformation , I think shocking the cells at 42°C water bath for exactly 90 seconds is much better.

Agree with everyone on the overnigh ligation. You can always transform part of your mixture after ligating for short time, and leave the rest overnight to transform the next day if no colonies observed.

On the transformation: do you have a positive control for transformation? I think 10ul is too much DNA for the cells, and definitely heat shock at 42C for at least 60sec (I also always do 90sec). Also, I usually only add ~250ul after the heat shock and 2min on ice before incubation at 37C. Alternatively you could spin down after the 37C incubation to concentrate the cells before you plate.

Thanks for all the prompt comments and advices.
I'll try overnight ligation and let you all know about the outcome.

lizzy & almost a doctor:
I'll try to extend the heatshock period to 60 sec; I hope I won't cause any harm to the cells

In fact, the amount of LB added prior to 1h shaking @ 37C was also one of my concern since I'll have about 600ml of them left after spread plate. I'd like to find out any good recommendation to break the pellet after spinning them down? Gently tapping on tube wall or...?

Thanks for your advices

Best,
Ayu

I agree with the amount of ligation product for transformation.
I use no more than 2 µL for 50 µL of bacteria.

For the plating, what I do is :

I add LB too, the final volume is 1 mL.
I plate 100 µL, and I centrifuge the 900 µL left, resuspend in 100 µL and plate it too. Then I get 10 times more colonies on this second plate.

Dear all,

I failed my transformation, again. No colony found on plate1 while satelites were spotted on plate2 (which I think could be due to contamination).

I run a gel on the ligation mixtures which I left on last two attempts (5 min ligation L3 & overnight L4), a ligation w/o insert (control insert-) L5 and the insert + vector (control ligase-) L6 (please ignore L2). Kindly refer to the attached gel image and comment.

Thank you.

BR,
Ayu

Ligation

I know it's a little stupid question, but it has happened here right now : could you check again that you are using the right antibiotic?
second, could you have a control where you cut your vector and re-ligate to be sure your ligase is working ?