EasyClone Normalized cDNA Library Construction Kit
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.
LITERATURE |
| User Manual |
RELATED PRODUCTS |
| EasyClone cDNA Library Construction Kit |
| EasyClone PCR Kit |
|
Manufactured by  |
|
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 approximately 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 |
ITEM #
| SIZE
| DESCRIPTION
| PRICE
| BUY
|
|---|---|---|---|---|
| P01011 | 20 Reactions |
EasyClone Normalized cDNA Library Construction Kit |
$2,350 |