1, To a tube add the following components:
 * 5 ul of  200 ng/ul acceptor vector
 * 10ul of Cre mix (1 ul Cre enzyme : 2 ul 10x Cre reaction buffer : 7 ul H2O)
 * 5 ul of  200 ng/ul donor plasmid

2, Incubate at 37 oC for 30 min.

3, Heat inactive reaction for10 min at 70 ˚C.

4, Transform bacteria immediately or store at -20 oC.

10x Cre reaction buffer

330 mM NaCl
500 mM Tris-HCl pH 7.5
100 mM MgCl2
20 % PEG

There are 3 possible stop codons but TAA is preferred because it is less prone to read-through than TAG and TGA. The efficiency of termination is increased by using 2 or 3 stop codons in series.

The Shine-Dalgarno (SD) sequence is required or translation initiation and is complementary to the 3′-end of the 16S ribosomal RNA. The efficiency of translation initiation at the start codon depends on the actual sequence. The concensus sequence is: 5′-TAAGGAGG-3′. It is positioned 4-14 nucleotides upstream the start codon with the optimal spacing being 8 nucleotides. To avoid formation of secondary structures (which reduces expression levels) this region should be rich in A residues.