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The second round of PCR used the product generated by the first round of PCR as its template and used new primer sets which generated restriction enzyme sites (Nhe I and Not I) at the very ends of the product for cloning.
The generated restriction fragment ends were repaired with T4 DNA Polymerase/dNTPs, and cloned into the SmaI site of a pUC19 vector [ 17].
The individual combination of enzymes was selected of the basis of computer generated restriction enzyme prediction from the BAC sequence (macro (Microsoft Word) available on request).
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First, genomic DNA PCR products of the complete SPRN ORF were digested directly with Ava1 endonuclease to generate restriction fragments amenable to PAGE sizing.
Optical mapping, a technique for generating restriction maps on a genomic scale [18], was performed on IBL 200 and IBL 122 by OpGen Inc. (Gaithersburg, MD, USA) using the restriction enzyme XbaI.
The genomic DNA was digested by blunt-end generating restriction endonuclease, RsaI (MBI Fermentas, USA).
Six micrograms of genomic DNA genomic were digested by blunt end – generating restriction endonuclease RsaI.
Others have attempted to generate restriction digestion independent Tag-seq using nebulisation [ 35].
Bioedit software was used to generate restriction maps from sequences obtained from SC and PS.
Briefly, EcoRI and MseI were used to generate restriction fragments that were selectively amplified by using a Cy-5 labeled Cy-5 labeled MsEco-Arimer.
These primers were used to amplify DNA fragments using Pure Taq Ready-to-Go PCR Beads (GE Healthcare, Pittsburgh, PA) followed by digestion with DdeI (New England Biolabs, Ipswich, MA) to generate restriction fragments.
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