Skip Main Navigation
NIH  


XGC

Sequencing Info

Other Species Collections

Related Links

Useful Links



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.