All donor fragments "TU + URA" were obtained by NotI digestion followed by gel purification.
Barcoded PCR products were then pooled and excess primers were extracted using gel purification (Promega gel cleanup kit).
Proteins were digested by proteinase K and DNA was purified by AccuPrep Gel Purification Kit K-30344, Bioneer).
Specific PCR product gel purification was followed by direct sequencing, without subcloning, in both directions.
Specific PCR product gel purification was followed by direct sequencing in both directions.
2) One can isolate sub-libraries with varied numbers of deleted building blocks by gel purification of digested SCRaMbLEd pools (Supplementary Fig. 2).
PCR products were pooled, cleaned-up, and purified using AMPure XP beads (Beckman-Coulter), quantified with picogreen (Thermo Scientific), followed by equimolar pooling and gel purification.
Yields of < or = 70% are obtained, and the siRNAs can be easily separated from any residual large dsRNA by a series of spin columns or gel purification.
TIANpure Mini Plasmid and Gel purification kits were from Tiangen (Beijing, China).
Nested PCR, gel purification, and vector ligation were carried out as described above.
The A-overhang added PCR product was purified using MinElute Gel purification kit (Qiagen, Germany).
