Designing PCR-Primers for LIC Cloning (Ligation independent cloning)
knowledge of the tagging strategy (N- or C-terminal tag ?)
Clone Manager software
Procedure
Delineate the coding sequence (full-length or partial sequence), which you want to amplify. Sense and antisense primers should have around 18 to 22 complementary bases, depending on the AT-content (there should be 9-10 Gs or Cs in the primer- cDNA complementary region). Both primers should ideally end with a 3’-GC clamp (two G or C bases).
In the antisense primer, add a STOP codon, if you want to create a protein with an N- terminal tag; omit the STOP codon, if you want to create a C-terminal tag.
Add the “LIC sequences” according to Fig. 1 to your sense/antisense primers. Take care to preserve the proper reading frame.
Fig. 1: Sequences to include in forward and reverse primers intended for LIC cloning. Codons representing the proper reading frame after transfer from pDNR-dual LIC are indicated. NNN indicates the start of the sequence complementary to your target sequence. Please remem- ber to use the antisense sequence in the reverse primer. Sequences to include in forward and reverse primers intended for LIC cloning.
With CloneManager, create a plasmid map of your planned vector. Baptise the new strain with a unique number of our strain collection (most cDNAs are sorted according to distinct topics; ask supervisor). Fill out a proper “Gentechnik” form for documenting your cloning procedure and indicate the new strain number on the form. Save the plasmid map on the FileServer using the strain number as file name and print a hardcopy. File both hardcopies, the plasmid map and the “Gentechnik” form, in the central folders on our common shelve.
Use CloneManager and your new plasmid map to design a proper control restriction digest for your putative clones. Think ahead about potential controls (empty pDNR- dual LIC, template vector encoding the cDNA of your PCR-amplicon, etc. ) for analyzing your clones.
If you have a correct clone (verified by restriction digest AND sequencing), streak out to obtain clear, single colonies from your clone, then streak a single colony on a new plate, and, next day, freeze down two aliquots of bacterial stocks. Place them in our - 80°C-strain collection and backup collection, respectively.