cloning 1052bp fragment - no colonies (Feb/10/2009 )

Hi,

I have deleted the lacZ gene from the pcDNA3.1(-)/myc-His/LacZ vector to be able to clone my gene it it. Has anyone tried this.I'm actually out of pcDNA3.1()-/myc-His/ vector and hence I'm using the control vector be deleting teh reperter gene. My insert is 1052bp long but I'm having trouble cloning it.

I have tried 1:3 and 1:5 vector:insert ratio i.e. 50ng vector but I'm not getting any colnies. I dd a control by transforming undigested plasmid and this results in several colonies. Thus, the transformation is fine. I had also run the T4 ligase with a marker and this results in a smear on the gel, hence, teh ligase is also working? Could my vectorbe a problem? If so, what control do I do to check for this.

Thanks
SF

SF_HK on Feb 10 2009, 04:18 PM said:

Hi,
It seems that the transformation efficiency is too bad. You will get much less colonies when transforming with the ligation mixture, compared to uncut plasmid.

I agree with WHR -- if your cells are competent, you should get way more than "several colonies" transforming with uncut plasmid -- closer to a lawn. If your cells are not competent enough, you'll have to be very lucky to get colonies transforming with a ligation mixture.

How did you prepare your competent cells? What is your transformation protocol? What selection do you have?

You could verify that the cells are fine by checking competency. Other thing will be to cut the LacZ out from your vector and then try to clone it back in to the same vector. This will show if the digestion was fine also if there is any other problem with the ligation and transformation.

What enzymes did you use to digest the plasmid and vector and for how long?

I meant I get lots of colonies on my plate when I transform using uncut plasmid.

I'm digesting my insert with XhoI and kpnI enzymes. I tried 1:4 vector to insert ratio and I get 10 very small colonies. Could the T4 ligase bffe be a problem since labmates didn't aliquot it and it has been freeze thawed more than 5 times.

SF_HK on Feb 13 2009, 07:14 AM said:

Did you check if the 10 colonies had the insert? All you need is 1 right colony.

Smell the T4 buffer, you will get the DTT smell. If you can smell it, then its fine. Or if you are not comfortable, get a new buffer.

I did a control reaction by adding ligase and single digested plasmid incubated at 16 deg for 16 hours. Followed by transformation. this did not give me any colonies.

Does hat mean that the ligase is not working.

I used the same liagese and liagated double digetsed plasmid(50ng) with my insert (200ng), 4deg 16hours, this gave 5 colonies but all empty?! So, what were those colonies if they didn't even have the self ligated vector?

Try getting a new ligase . sometimes that sort of things will do the trick.

Run all the ligation mixture on agarose, with the control for self-ligation and the insert alone (3 lanes), extract out the band in the lane of ligation mixture (which one is different from the 2 remaining lanes and if you can see it), then purify and transform to competent cell (should use more than 10% volume of competent cell; in my case, i use electroporation: 4ul ligation mixture + 30ul competent cell). You will get not much colonies but most of them harboring the correct one. I though about it some months ago and I tried it recently. So happy that I succeed in ligating the 8kb insert into 7kb vector by blunt-end ligation (I took me months with troubles until this time ^^). If your problem doesn't lie in the ligase or competent cell matters, you should try my strategy . Good luck!

That is an interesing way. But won't the ligation reaction give a smear when running on agarose gel? One silly question, you woulb be adding the loading dye to the ligation reaction before loading to the gel?