Multigene cloning - Looking for a cheap solution - (Jul/27/2008 )
I need to insert 5 DNA fragments into 1 vector. It is impossible to find restriction sites to do this.
I found Gateway from Invitrogen is a good solution. However, it is too expensive.
Any suggestion for a cheaper solution?
Yeah you can do it, 6 way ligation. Just use normal NEB T4 ligase buffer and incubate overnight at 16 Celsius (Do not use quick ligase of any sort) Use colony PCR to amplfy over the junctions between inserts. I would recommend you screen by colony PCR with the aid of a multichannel pipette at least 200 colonies.
If you want you can use SLIC method. I have not used this method before, but it sounds interesting.
Are you trying to make five clones, one with each inert in it, or one clone with all five in it?
Do you know the sequences of the fragments?
I also need to insert 3 inserts into a vector. I am planning to use the SLIC method. This paper may be helpful:
Nature Methods - 4, 251 - 256 (2007)
Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC
Mamie Z Li & Stephen J Elledge
Hi Lotus, if it is not too much trouble could you be so kind as to report back the results of the SLIC method? I would like to hear you personal experience with this method