Cloning with linkers? - (May/24/2007 )
I have to clone an insert (5'BamHI-Insert-EcoRI 3') into a vector containing XbaI, BamHI and 2x SalI at the cloning site.
I thought the simplest way would be to amplify the insert (2kb) with PCR primers to add a SalI site at 3' of the insert, and cut-ligate it into the vector.
However I tried the Vector NTI cloning design program, and it came up with the suggestion of cutting vector and insert with appropriate enzymes (XbaI+SalI, and BamHI+EcoRI, respectively), fill completely the overhanging strands, and ligate XbaI to all termini before ligating the insert to the vector. The correct orientation would be verified through restriction analysis.
This approach sounds more complicated, but avoids the PCR step, and may be usefull for another cloning I need to do, in which the cloning site of the receiving vector has no convenient restrictions sites.
My question is the following:
- Is the idea of using linkers for cloning is just very bad, and I would better stick to PCR cloning?
- I guess I should use Klenow to fill the overhanging strands... Is there anything special to take into consideration for the subsequent cloning of the insert and vector (like phosphorylation, dephosphorylation)
- Does anyone have any experience with linkers blunt end ligations? Which concentration for the oligos relative to the fragment, etc?
- Should the linker oligos have some additional residues to facilitate restriction? I guess 6 nucleotides are a minimum, as per this NEB page
If you design the linkers properly, you don't need to cut them. Anneal a pair of oligos of different lengths to give the correct 3' or 5' overhang. Make sure the 5' end is phosphoylated when you order them.
Personally, I wouldn't do it this way, but I'm sure it can be made to work. I'd choose the PCR approach.
SalI is a widely denigrated enzyme -- you might want to choose an alternative.
Just a thought -- how about an EcoRI / XbaI adapter.
That's a good idea, and it simplifies things!
I think in the end I will go for the PCR method... As a side question, when you use primers to add restrictions sites, do you use a PCR protocol with a first step at the right melting temperature, and the other annealing steps at the full length primer melting temperature? How long do you set up the first annealing step?
Ok, I can insert BamHI on the 3' end with a primer (and remove the 5' BamHI site with another primer), and use BamHI to insert the PCR product to the vector
Thanks for the input!
I would go with the PCR method.
I would like to add the concept of compatible cohesive ends. Several restriction enzyme produce the identical sticky ends and thus these ends are able to ligate to each other.
For example. XbaI, NheI and SpeI all make sticky ends that are indentical and thus cross compatible.
A list of compatible cohesive ends can be found on NEB's website, under restriction enzymes.
As for the annealing question,
I use only one annealing temperature, the annealing temperature of the primer sequence that binds to the template. I do not consider the restriction site sequence in the calculations.
Yes, this does ignore the binding of the RE site sequence in later cycles, but one can consider this an inverted pseudo touch down PCR. Touchdown PCR is a method to increase yields, it does so by lowering the annealing temperature in later cycles of the PCR. Yeilds increase and specificity decrease, but since most of the template (produce earlier in the PCR reaction) is short and percise, so no secondary bands are produced. And thus yeilds are not compromised.
In our invert case, the annealing tempearture stays the same but the primer melting temperature increases
That's a good point, but my choice of restriction enzymes is limited for cutting the insert and the receiving plasmid. BTW, here is the NEB list of compatible cohesive ends.
Thanks for the tip, that's very helpfull (as the added explanation!)