Table 1 Summary of optimal conditions for DNA assembly with AFEAP cloning
From: AFEAP cloning: a precise and efficient method for large DNA sequence assembly
Parameters | Tested conditions | Optimal conditions |
|---|---|---|
Overhang length | ranges from 0 to 20 nucleotides | 5–8 nucleotides |
Size of DNA fragments | 5.5, 8.0, 15, 20, and 30 kb | Decreased with the increase of DNA size |
Overhangs designed as 5′ end of G/C or A/T | overhangs designed as 5′ end of G/C or A/T | G/C |
Ligase treatment | T4 DNA ligase treated or not | Ligase treated |
Ligation ratio (Shorter to longer) | 1:6; 1:3; 1:1; 3:1; 6:1; 10:1, 15:1; and 20:1 | 10:1 |
Transformation conditions | Chemical, electroporation | Chemical transformation |
DNA fragment number | 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 | Decreased with the increase of fragments number |