Correct orientation of the insert - (Feb/12/2010 )

Hi all,

I'd like to share my problem with you, and ask you for a piece of advice.

I am trying to clone one 1,7kb insert into about 14kb plasmid. The insert comes from a TOPO, digested out with Xho I and Sal I. It has to be inserted into the vector, which is cut only by Sal I. Now there is the tricky part, Sal I is unique in the vector, so it is linearized. On the other hand, digestion of the insert leaves compatible ends which can bind in any orientation into the vector. I am getting some colonies, sometimes more, sometimes less. Some of them are positive (they have the insert), but so far none of them has it in the correct orientation.
I think that my maps are correct, I triple-checked them, the TOPO containing the insert has been sequenced, the ligation is OK (both + and - controls are fine), enzymes are OK (checked with control plasmids) just I am not getting the positive colonies with correct insert orientation!!
If anyone has any idea, please share it with me. I've tried the cloning 5 times so far, without any success.
Any suggestion is welcomed!
Thanks, in advance.

Best

If I understand it correctly your insert can be inserted either in the correct way or the reversed way?

If this is true, then there is nothing much you can do: it all depends on "luck" wether the insert is placed correct or in the reverse way.
Unless you can change the insert of plasmid at one site by adding, removing some nucleotides to get sticky ends that are not the same.

And you tried 5 times, well let me tell you something: I once tried 27 times before I had a good insert:p

Its not all that easy or fast when working with things like this.

Its easier of the ends are not the same and thus you can have a reversed insert.

pito on Feb 12 2010, 09:06 PM said:

Thanks a lot!
I thought that I'm doing something wrong.
Also I was wondering if there is some trick to force the insert to orientate (as I don't have much experience in cloning), so that's why I posted the q.
I'm trying again today.

MacJoseph on Feb 15 2010, 09:45 AM said:

As it happens there ARE a few thing that can help:
1) Ignore the construct! make it think that you do not need it in the correct orientation, sometimes it works!
2) Use the Force! (works, always, but u must be a jedi Knight!)
.......
3) I'm clearly joking (but number 1 sometimes seems to be true) but i was wondering one thing: are you checking ALL your positive colonies? because once you have all things done the right way doing everything time and again til it works is like testing the 1000 colonies you have the first time, but u already have everything and do not waste competent cells and money.
thats just my opinion but it's tested that sometimes (sadly...rarely ) even it works!
bye Fiz

Sometimes the destination plasmid transcribes the gene inserted, and, if the gene is toxic, you will not see the insertion event, since cells with successful ligation will die. You may want to think about changing to a promoter which can be completely inactivated (T7 promoter with no T7 DNA polymerase gene in the cell, e.g.).

Cloning for the correct orientation is far far easier if you use two distinct REs rather than one.
I would recommend that you rethink the cloning strategy.

If you continue to use single RE cloning, then you can become more efficient in your analysis of clones with colony pcr, using one forward primer on the vector and a second reverse primer on the insert. Amplification will occur only with the insert present and in the correct orientation.

I would agree with phage here re the point to change your strategy into one with different 5' and 3' restriction sites ...

From my personal experience: i have just abandoned trying to clone a similar size insert (1.8kB) into a similar sized vector (13kB) with same sticky ends on 5' and 3' end (EcoRI). My insert was also only ever inserting the wrong way around (and that not very efficiently). I ended up screening loads (and loads and loads) of colonies by colony PCR using one primer in the insert and one in the vector (as phage described).

This way I did manage to find a few positive colonies in the end, but somehow the bugs managed to scramble around the plasmid DNA so much in later steps, that I never succesfully managed to grow even the smallest miniprep I tried growing at lower temperatures (25C rather than 37C), different competent cells (Sure cells, TOP10, DH5aplha), growing for <5hrs rather than O/N, etc.etc.etc.

To summarise: I am now going to try different sticky ends. As for the potential toxicity of the gene... I am not sure how to solve this. Posted a while ago to see if anyone has any experience with the competent cells by NEB that supposedly allow for cloning of toxic genes, e.g. 5-alpha F'Iq and Turbo strains, but got no replies unfortunately...

Good luck

As the insert can orient itself in any direction, they somehow prefer the orientation which is oppositeof what we would like it. But as there is some probability involved, you mighthave to screen a lot of colonies before you could get the right orientation. I had struggled once with such a problem and ended up analysing more than 60 clones.

phage434 on Feb 15 2010, 03:20 PM said:

Thank you all for the suggestions.
About the promoter part: the promoter is as important as the reporter gene. It's a mammalian neuronal promoter... Previously (couple of months ago) another gene was cloned under the same promoter.
About the single digestion of the vector: again, the best option to stay in the reading frame of the promoter is the Sal I.
The cells used: DH5 alpha from Invitrogen. I might try some others, or change the strategy.

Thanks again to all of you.