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[Construction of cDNA library from human colorectal cancer cell HRT-18]
1Cancer Research Institute, Xiangya School of Medical, Central South University, Changsha 410078, China.
Objective:
To construct a cDNA library from human colorectal cancer cell HRT-18.
Methods:
The total RNA was extracted from human CRC (colorectal cancer) cell HRT-18 and the mRNA was isolated from the total RNA by MagneSphere technique, and then the first-strand cDNA was synthesized with oligo (dT) primer containing sfi I site while the double-strand cDNA was amplified through LD-PCR (long-distance PCR) by SMART technique. The double strand cDNA was digested by sfi I ( I A & I B) restriction enzyme before cDNA size fractionation, and the double-strand cDNA fractionated was ligated into the lambdaTripIEX2 vector and then was packaged into lambda phage in vitro.
Results:
The unamplified human CRC cell HRT-18 cDNA library consisted of 6. 5 x 10(6) independent clones, recombinants clones was more than 96%. The titer of the amplified cDNA library was 1.1 x 10(9) pfu/ml and the average insert of the recombinants was 1.1 kb.
Conclusion:
The quality of the constructed cDNA library from human CRC cell HRT-18 is excellent and it is helpful to screen CRC specific antigen.
