Does the In-Fusion Cloning method introduce errors into the sequence?

We have not seen any base slippage, base addition, or base deletion with the In?Fusion Cloning enzyme. We have cloned and sequenced over 4,000 separate clones and various human open reading frames subsequent to In?Fusion Cloning, and have rarely seen any evidence of errors at the cloning junctions (<2%). Most of the sequence errors that we have come across are clearly due to errors in primer [...]

What is Cloning Enhancer (CE)?

Cloning Enhancer, or CE, is a proprietary enzyme mix for removing background plasmid DNA and PCR residue, thus eliminating the need for additional purification of PCR-amplified DNA prior to the In?Fusion reaction (see details in the In?Fusion Snap Assembly User Manual). CE is available as a separate item. Use of CE is only appropriate if PCR amplification generates a single PCR fragment of the [...]

What is the difference between In-Fusion Snap Assembly and In-Fusion Snap Assembly EcoDry?

In-Fusion Snap Assembly includes liquid In?Fusion Snap Assembly Master Mix, whereas In?Fusion Snap Assembly EcoDry includes pre-aliquoted, lyophilized In?Fusion Snap Assembly Master Mix. The EcoDry kit minimizes handling and is stored at room temperature. Both liquid and lyophilized master mixes have a cloning reaction of 15 min. In-Fusion Cloning [...]

How should cells be dissociated from RetroNectin-coated plates?

For strongly adherent cells, like fibroblasts, use trypsin-EDTA (without Ca2+ and Mg2+) to dissociate cells. For weakly adherent (or suspension) cells, use a 0.02% EDTA/PBS solution following the protocol below. Use trypsin to remove these cells only if the EDTA/PBS treatment is unsuccessful; trypsin may damage these cells. 1. Following transfection, transfer the supernatant from the plate to a [...]

After transduction of cells using RetroNectin reagent, what method do you recommend to resuspend the cells?

For cell lines such as HL60 cells and K562 cells, we have verified that cells can be easily collected by pipetting from the RetroNectin reagent-coated dish. However, for several other cell lines, it may be difficult to collect the cells by pipetting without trypsin treatment. In general, if cells cannot be collected by pipetting, we recommend collecting cells by trypsin treatment 24 hours after [...]