酵母双杂交系统EasyClone normalized cDNA library construct-北京东方安诺生化科技有限公司

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酵母双杂交系统EasyClone normalized cDNA library construction package
Fast and Easy Synthesis of Highly Complex cDNAs

Directional cloning of cDNAs

The EasyClone Normalized cDNA Library Construction Kit is designed to synthesize full-length-enriched
double stranded （ds） cDNA from total or polyA+ RNA, followed by normalization of amplified cDNA and subcloning into an appropriate library vector. The kit is based on a novel technology utilizing the specific features of MMLV-based reverse transcriptase to create full length enriched double stranded cDNA from total or polyA+ RNA samples. cDNAs are amplified by representational PCR and cloned directionally into library vectors for use with yeast two-hybrid systems such as Dualsystems' DUALmembrane, DUALhybrid, and DUALhunter protein interaction screening kits.

No extensive know-how in library construction is needed - the kit supplies all reagents needed for library construction and the detailed manual will guide you through all steps of construction.

Construction of normalized cDNA libraries

Normalization is the process of removing abundant RNAs from the starting material. Normalized cDNA libraries contain approximay equal amounts of all RNAs and therefore increase the occurrence of rare proteins. This significantly increases the quality of yeast two-hybrid screens.

Figure 1: qPCR of normalized and not normalized human spleen cDNA. For Actin, a decrease

of 75.8% was reached. For hGAPDH, the decrease was 60.6%. The amount of these two high

abundant cDNAs was lowered, increasing the quality of the cDNA library.

KIT CONTENTS - Box 1
5X first strand buffer	80 µl
DTT （20 mM）	30 µl
10X dNTP mix （10 mM each）	110 µl
PlugOligo-3M adapter （15 µM）	25 µl
CDS-3M adapter （10 µM）	25 µl
EasyClone reverse transcriptase	20 µl
IP-solution	130 µl
Control RNA template （0.5 µg/µl）	15 µl
Sterile RNase free water	1.8 ml

KIT CONTENTS - Box 2
50X EasyClone polymerase mix	50 µl
10X EasyClone buffer	300 µl
PCR Primer M1 （10 µM）	170 µl
Sterile RNase free water	1.8 ml
Control amplified cDNA sample 1	25 µl
Control amplified cDNA sample 2	25 µl

KIT CONTENTS - Box 3
NORMALIZER enzyme, lyophilized	50 units
NORMALIZER storage buffer （50 mM Tris-HCl pH 8.0）	70 µl
4x Hybridization buffer （200 mM Hepes, pH 7.5, 2 M NaCl）	70 µl
2x NORMALIZER master buffer （100 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 2 mM DTT）	250 µl
NORMALIZER stop solution （5 mM EDTA）	500 µl
NORMALIZER control template, 100ng/μl	20 µl
ACTB plasmid （100 ng/μl）	50 µl
UBC plasmid （100 ng/μl）	50 µl

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