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pCS107 Vector Library Method
Libraries in the pCS107 vector were constructed in the laboratoy of Bruce Blumberg of University of California, Irvine. Briefly, the method is as follows:
- Total RNA was prepared from homogenized tissues using Trizol (Invitrogen). Poly A+ RNA was isolated from total RNA using Oligotex (Qiagen).
- First strand cDNA was synthesized using Stratagenes’s StrataScriptTM reverse transcriptase and anchored oligo-dT primers containing an Xho I restriction site. The primer sequence was 5’-ACTAGTGCGGCCGCCTAGGCCTCGAGdT(18)-3'
- Second strand cDNA was synthesized using DNA Polymerase I and Rnase H. DNA Polymerase I.
- The double stranded cDNA was blunt-ended using Pfu DNA polymerase, ligated to Eco RI adapters, treated with T4 polynucleotide kinase, and finally digested with Xho I.
- cDNAs were passed over Sepharose CL-2B column and fragments larger than 1000 bp were collected.
- The size-selected cDNAs were ligated into Eco RI-Xho I-digested pCS107 that had been phosphotased.
- Libraries contained between 4 x 105 and 1 x 106 recombinants, and were stored at –80o C in SOC media containing 10% DMSO.
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