Library-10000 Sequencing
Library-10000 Sequencing
PCR read-off microarrays: Extension reaction mix (200 uL) without primers was prepared according to a Promega standard protocol using a PCR Master Mix (Promega; 25 U/mL Taq Polymerase, 200 uM dNTP, 1.5 mM MgCl2) was loaded onto the microarray using an Agilent hybridization cover slide. The first extension step was carried out at  55C for 16 h (overnight).  Hereafter, the reaction mixture was removed using a pipette and fresh PCR reaction mix (200 uL) with Solexa-primer-1 and 2 (0.1 uM) were loaded onto the microarray and a standard PCR cycle was set up in a GeneMachines Hyb4 automated hybridizer [40 cycles; denaturation at 94C for 30 s, annealing at 65C for 1 min, extension at 70C for 1 min]. In addition, an initial 3 min denaturation step at 94C and a final 15 min extension step at 70C were carried out. Immediately after the PCR had finished the reaction mix was collected using a pipette and the microarray washed with H2O (3 x 50 uL). The aqueous fractions were pooled together and concentrated by speed vac followed by purification by preparative DNA gel electrophoresis (dsDNA-10,000: 1.65 ug, 40% isolated yield). PCR in solution: The purified product (250 ng) from the PCR read-off microarrays were used as templates in another round of PCR with Solexa-primer-1 and 2 (1 uM) in a 1 x PCR Master Mix (200 uL) in a Techne TC-312 PCR cycler with the same cycle as on microarray. After PCR the samples were concentrated by speed vac followed by purification by preparative DNA gel electrophoresis (dsDNA-10,000-2: 16.8 ug, 30% isolated yield). This sample is not extracted from any biosample material as it is a synthetic PNA library.
Experimental Factor: INDIVIDUAL
Synthetic 10000 DNA library