3 replies to this topic

[Sandy Fowler]

Dear all,

Hi, I'm very new to cloning and I have been having trouble figuring out why I have colonies growing on LB-Amp plate but when I do minipreps of the colonies and digest them with the enzymes that created the sticky ends for the plasmid and the insert, no insert can be found.

I had a control (Just vector in the ligation with no insert), and I transformed that into cells and I had no colonies. I have about 15 colonies on my vector+insert plate.

I did not run a gel for a ligation reactions, I just transformed the ligation mixture (10ul) into One Shot Top10 E. coli cells by Invitrogen.

The transformation protocol is as followed:

- thaw cells on ice
- add 10ul of DNA/ligation mixture
- incubate on ice for 30 minutes
- heat shock at 37C for 5 minutes
- add 700ul LB and grow at 37C for 1 hour
- spin down cells and resuspended with 200ul of LB
- plate on LB+Amp agar plates

Ampicillin stock concentration is 100mg/ml and I added 1:1000 for making the LB plates.

Is there something wrong with my transformation protocol? Or is it my ligation?

Vector(5.7kb) and PCR insert (1.5kb) were digested with EcoRI and XhoI for 3 hours and the vector was treated with CIP for 1 hour.

Also, ligation protocol:

2ul vector
7ul PCR insert
1ul T4 DNA ligase from NEB
2ul 10x buffer
8ul dH2O

incubate at RT overnight

Any input would be appreciated.

Thank you very much

[bob1]

OK, the protocols look fairly standard (apart for a few bits, but I'll come to those), but there are a few things that you need to tell us - How big a volume of cells did you transform?  How much total DNA did you use for the vector and insert? - volumes aren't really helpful unless we also know the concentration.  Did you clean up the vector and insert after digesting?  Is the insert PCR generated or are you subcloning from another vector?  Did you run digestion controls so that you know your digestion is working? Was the ligation buffer fresh?

Check the manual for the Top10 cells, it will probably say to heat shock for 1-2 minutes at 42 deg rather than 37, which is a non-standard temperature.

You would usually get more than 15 colonies on a vector+insert plate if the ligation worked, did you have a transformation control (uncut plasmid, usually something small like one of the pUC series that you transform so you know that the transformation worked)?

Ligations are usually done at room temp for 30 min-1 hour, 16 degrees overnight or 4 degrees for a day or so, RT overnight might have caused a problem.

As you are creating non-compatible sticky ends you shouldn't need to use CIAP (just use appropriate transformation controls).  Phosphatase treating often causes more problems than it solves, as it is very very hard to remove and incredibly heat stable - the manufacturers lied when they said it could be heat inactivated at 75 for 30 min, it will still be working.

See also this page for somethings to think about.

If you are worried about running out of competent cells - don't worry, you can easily and cheaply make some more following this protocol.

[Sandy Fowler]

Dear bob1,

I transformed 10ul of the ligation into 50ul of Top10 cells. I did not quantify the vector nor the PCR insert because the nanodrop in the lab is not very reliable.

Also, I purified the insert in two ways: spin column and UV gel extraction. I got 5 colonies from the spin column purified PCR insert ligation and 15 from the UV gel extracted PCR insert ligation.

Thank you so much for your input. I think I am going to ask around to see if another nanodrop is available and redo the ligation and transformation with your suggestions

[bob1]

Good luck, cloning is definitely a technique that needs a bit of luck as it is often tricky and unreliable.

Note that molar ratios of vector:insert (see links in my above post) are usually used for these sorts of things, and it is often found that it will work at one particular ratio,  but not others, so try a few.