Overview: cDNA stands for complementary DNA or copy DNA and can be single-stranded or double-stranded. cDNA is synthesized in vitro from a mRNA template using reverse transcriptase. The resulted cDNA is single-stranded. This process is called reverse transcription (RT) or first strand cDNA synthesis. In some experiments such as microarray, double-stranded cDNA is required and can be produced by another round of DNA synthesis following the first strand synthesis. The purpose of converting mRNA to cDNA is mainly for the analysis of the template mRNA because DNA is much stable than RNA. Once mRNA is converted to cDNA, the cDNA can be used for RT-PCR, as probe for expression analysis and for cloning of the mRNA sequence.
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Protocols
- cDNA Production and Cloning ( Dr. Bart Frank, Arthritis and Immunology Program, Oklahoma Medical Research Foundation)
Produce cDNA and subsequent clone it into vector. Protocol includes: 1st Strand cDNA Synthesis
Determine the efficiency of first strand cDNA synthesis
Second Strand cDNA Synthesis
Cleave 5' Overhanging Ends
Methylate to Protect EcoRI Sites in cDNA
Ligate to Linkers
Ligate cDNA to Vector
http://omrf.ouhsc.edu/~frank/CDNA.html
Added: Thu Sep 01 2005, Hits: 6324, Reviews: 0 Write review Cached -
cDNA Library Construction
(Michael Blaber's Lab)
Very useful guide and protocol for cDNA library construction
http://wine1.sb.fsu.edu/bch5425/lect27/lect27.htm
Added: Tue May 14 2002, Hits: 3277, Reviews: 0 Write review Cached -
cDNA Synthesis
(Cungui Mao)
http://www.musc.edu/bcmb/ceramide/protocols/0043.h...
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cDNA Synthesis
(Promega)
Provides detailed procedures for construction of cDNA library, including first strand and double-stranded cDNA synthesis from mRNA, and subsequent ligation into a suitable vector.
http://www.promega.com/tbs/tm038/Tm038.html
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cDNA/AFLP
(CWB Bachem)
The protocol which follows gives a step-by-step description of cDNA-AFLP, a method for visualisation of differential gene expression
http://www.dpw.wau.nl/pv/staff/aflp.htm
Added: Tue May 14 2002, Hits: 1162, Reviews: 0 Write review Cached -
Differential cDNA Screening
(Gerard Lazo)
cDNA library construction and differential screening
http://wheat.pw.usda.gov/~lazo/methods/misc/Talbot...
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ESTs from Phage Plaques
(the WHO/UNDP/World Bank Schistosoma Genome Network)
This protocol uses PCR to amplify phage insert for sequencing and EST production.
http://www.nhm.ac.uk/hosted_sites/schisto/protocol...
Added: Tue May 14 2002, Hits: 565, Reviews: 0 Write review Cached -
ESTs from Phage Plaques
(the WHO/UNDP/World Bank (WHO)
Schistosoma Genome Network)
A slightly different protocol from the above one.
http://www.nhm.ac.uk/hosted_sites/schisto/protocol...
Added: Tue May 14 2002, Hits: 345, Reviews: 0 Write review Cached -
Library cDNA Synthesis
(Dr. Simon Dawson, Department of Biochemistry, University of Nottingham Medical School)
Dealing with first strand and second cDNA synthesis for library construction.
http://www.nottingham.ac.uk/~mbzspd/methods/Librar...
Added: Sat Apr 10 2004, Hits: 1278, Reviews: 0 Write review Cached -
Protocol for First-strand cDNA Synthesis
(Fermentas)
http://www.fermentas.com/TechInfo/ModifyingEnzymes...
Added: Tue May 14 2002, Hits: 3885, Reviews: 0 Write review Cached