( Library ...)Constructing Directional cDNA Libraries from Limited Amounts of RNA

Library Name

Class 1

Class 2

Class 3

Class 4

Class 5

HepG2 Lambda Library

61%

4%

8%

12%

15%

HepG2 PCR library

48%

1%

7%

19%

15%

Conclusions

Stratagenes new PCR Library Construction Kit allows efficient and directional cloning of cDNA inserts into the pCMV-PCR vector. The kit is complete and can be used to generate libraries from purified mRNA as well as limited amounts of total RNA. It is particularly useful in situations where samples are difficult to obtain, such as LCM, early development stages, and any specially isolated cell population. The kit combines PCR technology for amplifying cDNA with the LIC technique for cloning PCR products: we successfully constructed numerous libraries, including an ovarian tumor library from human LCM tissue. The library inserts were confirmed by PCR and sequence analysis. The HepG2 library comparison clearly shows that no artifacts are introduced due to the PCR-based method (i.e., no particular gene is over represented), and that the PCR-generated method is an efficient way to
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