Cloning using adaptors-->converting RE site - (Aug/16/2007 )
Here is my dilemma:
I have an 8kb dna fragment that I would like to remove from one plasmid (NotI RE site) and clone into another vector which does not have a NotI site. I was thinking of designing an adaptor to convert the NotI sticky ends of the fragment into XhoI sticky ends.
It seems like a good idea in theory, but I don't really know how to carry it out.
If I order two partially complementary oligos, that when annealed to each other create an adaptor with one side having a NotI sticky end and the other the XhoI sticky end, how do I go about ligating it to my fragment?
1. After I anneal the adaptor (I assume I would just mix the two stands 1:1, heat to 94C and anneal at 55C or so for a few minutes) do I need to purify the ds-product? If so how?
2. Before I anneal the adaptor to the DNA fragment do I need to phosphorilate it? Or are all the necessary phosphates there already because it was never de-phosphorilated?
3. In what ratio do I mix the fragment and the adaptor? I assume I would need to gel purify the fragment after annealing the adaptors, before ligating it into my target vector...
any help or alternate suggestions much appreciated!
or if you know of a good protocol that deals with this please let me know, I haven't been able to find anything useful.
1. After I anneal the adaptor (I assume I would just mix the two stands 1:1, heat to 94C and anneal at 55C or so for a few minutes) do I need to purify the ds-product? If so how?
No. There is no need to purify the dsDNA.
Unless specifically ordered by the customer, your primers will come to use in a dephosphorylated state. This DNA was chemcially sythesised, thus 5' phophorylation does not occur. YOu can buy phophorylated adaptors/primers
As for phosphorylating the insert it depends. I will talk about that later.
As high as you can. The ligation efficiency is notoriously low. I would use 13ul of 20uM of adaptor + 5ul of cut plamids DNA.
As for phophorylating your adaptor....
well you can leave it.
Ie cut your plasmid with Not. Add your adaptor, which is designed to remove the Not site and replace it with Xho. After ligation, cut your ligation mix with Not again, to remove any plasmid which did not ligate to the adaptor. This method is simple but messy.
Alternatively, you can by buy or make a phophorylated adaptor. Cut and dephosphorylate your vector. Ligate the two and screen for colonies that have accepted the adaptor. Method is easier, however your adaptor can only carry one restriction site. As adaptor concaterms are a real problem.
Looking at your situation, the second method is the most straight forward.
My approach would be to add a NotI restriction site to the recipient vector using quikchange, which is a very efficient PCR-based method for site directed mutagenesis.
Thank you for your suggestions!
Perneseblue, I'm not sure what you mean my this:
If I cut the ligation mix after ligation, will it not remove the adaptor I just ligated? I don't think the NotI site will be lost with the ligation of the adapter... Maybe I am not undersanding what you mean.
I was thinking of designing two ss oligos that when annealed form a duplex:
5'- TTGAGG
CCGCCGGC-5'
This way when the NotI sticky end anneals to my DNA fragment with complementary ends, the NotI site will be restored, but now there will be new XhoI sticky ends.
After thinking some more about this, I wonder if it is less trouble to just fill in the sticky ends and blunt clone the fragment...
bitesizebio_guy My target vector was constructed by someone in another lab, so I don't know enough about its sequence to design primers. I guess I could ask them, but I've had some bad luck with long PCR in the past (this vector is 6kb long) so I'd rather stay away from it. Also I'd worry about PCR introducing mistakes into the vector.
Sorry for the break down in communication. 
I got confused which restriction site was present in the vector. Please subtitude XhoI when ever I mentioned Not
Hmm... do you want to preserve the NotI site or get rid of it?
But assuming you do want to keep the Not I site, I have the below suggestion.
5' - GGCC GCCTAGG
CGGATCC AGCT -5'
You can then conduct an XhoI kill digest to kill any vector that has self-ligated
Hmm... will more thought, I think you should keep the adaptor to insert to vector ratio a 2:1:1 upto 3:1:1
Flooding as I ealier suggested will not be the best thing to do in this situation.
Alternative suggestion (aside from blunt end cloning), would be to change the XhoI site in the vector to a NotI site first with an adaptor. Once done, the insert goes in nicely.
You can leave both vector and insert phophorylated. The adaptor is left dephosphorylated. Throw the whole mix in. Do the kill digest to remove self ligated vector. Screen 72 to 96 colonies by colony PCR.