What are the recommended insert-to-vector molar ratios for In-Fusion Cloning?
What are the recommended insert-to-vector molar ratios for In-Fusion Cloning?
In-FusionCloning uses a very robust enzyme, and allows highly efficient cloning in most situations. General recommendations on insert/vector quantities are included in all current In-Fusion Cloning user manuals.
-To ensure optimal results under standard conditions, or when performing single- or multiple-fragment cloning, use an insert-to-vector ratio of 2:1.
*The molar ratio of each of the multiple inserts should be 2:1 with regards to the linearized, purified vector. The molar ratio of two inserts with one vector should be 2:2:1.
*To calculate the required amount of each of the DNA fragments, use no less than 20 ng of the smallest insert and calculate the quantities of the rest of the fragments accordingly, maintaining the 2:1 insert to vector molar ratio. Each of the inserts should be calculated at the 2:1 molar ratio with regard to the vector.
-For cloning of small DNA fragments (between 150 and 350 bp), the suggested insert-to-vector molar ratio is 3–5:1.
-For cloning of short synthetic oligos (between 50 bp and 150 bp), the suggested oligo-to-vector molar ratio is 5–15:1. Depending on the oligo length, the optimal molar ratio must be determined empirically.
*Non-phosphorylated oligonucleotides are compatible with In-FusionCloning. However, 3' exonuclease activity in the In-Fusion enzyme mix requires terminal 3' OH groups.
Use our online Molar Ratio Calculator to calculate specific insert-to-vector quantities based on molar ratios, insert length (bp), and vector length (bp).