AflII NEB#R0520

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	For AflII and AvaII digest of cloned DNA building blocks, the RCA reaction was diluted 4-fold with water. Digest formulations and incubation temperatures for BsrDI, AflII and AvaII (NEB) are given in Supplementary Table S2. For any digest, a mastermix of buffer, enzyme and BSA (if applicable) was created, then added to the diluted RCA reactions. After addition of enzyme mastermix to diluted RCA products, digest reaction was allowed to proceed for 4 h to ensure complete digestion, followed by heat inactivation for 20 min and finally 10-fold dilution with water. The diluted sample was analysed by capillary electrophoresis on the Fragment Analyzer instrument for verification of digest pattern.

Protocol tips

For AflII and AvaII digest of cloned DNA building blocks, the RCA reaction was diluted 4-fold with water. Digest formulations and incubation temperatures for BsrDI, AflII and AvaII (NEB) are given in Supplementary Table S2. For any digest, a mastermix of buffer, enzyme and BSA (if applicable) was created, then added to the diluted RCA reactions. After addition of enzyme mastermix to diluted RCA products, digest reaction was allowed to proceed for 4 h to ensure complete digestion, followed by heat inactivation for 20 min and finally 10-fold dilution with water. The diluted sample was analysed by capillary electrophoresis on the Fragment Analyzer instrument for verification of digest pattern.

Publication protocol

"Amplification of plasmid constructs from E. coli cultures was done by multiply-primed RCA (15) using commercial buffers and mastermix (MCLAB). Two microliters of overnight E. coli culture was added to 5 µl of lysis buffer, then incubated at 96°C for 5 min. After addition of 5 µl mastermix containing the mesophilic phi29 polymerase and random hexamers (15), RCA reaction was allowed to proceed for 16 h at 30°C. For BsrDI digest of assemblies, the RCA reaction was diluted 2-fold with water. For AflII and AvaII digest of cloned DNA building blocks, the RCA reaction was diluted 4-fold with water.

Digest formulations and incubation temperatures for BsrDI, AflII and AvaII (NEB) are given in Supplementary Table S2. For any digest, a mastermix of buffer, enzyme and BSA (if applicable) was created, then added to the diluted RCA reactions. After addition of enzyme mastermix to diluted RCA products, digest reaction was allowed to proceed for 4 h to ensure complete digestion, followed by heat inactivation for 20 min and finally 10-fold dilution with water. The diluted sample was analysed by capillary electrophoresis on the Fragment Analyzer instrument for verification of digest pattern."

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