Marligen cDNA Library Technology

Marligen prepare high quality standard, large-insert, normalized, subtracted , micro-quantity and nano-quantity cDNA libraries using their proprietary technology.

Standard, Micro-quantity and Nano-quantity Libraries

First strand cDNA is made from mRNA that is primed using oligo dT. After the second strand is synthesized, the double stranded cDNA is size fractionated, cloned directionally into a Marligen Express I vector, then transformed into T1 phage resistant E. coli.  Marligen scientist’s obtain directionality by using the Not I and EcoRV (blunt end) sites within the Express I vector.  However, other restriction sites are also available. See the tables below for material requirements and guaranteed yields.

Large-insert libraries

Large insert libraries are made similarly to standard libraries except that extra processes are utilized in the size fractionation step to recover larger cDNA fragments. For large-insert libraries, Marligen require at least  10 ug of mRNA, 2 mg of total RNA, 2x108 cells or 200 mg of tissue, and Marligen guarantee an average insert size of 3 kb.

Sample Requirements

Guaranteed Results

Normalized Libraries

Normalized cDNA libraries are produced from customers' custom cDNA  libraries (i.e., standard libraries, microquantity libraries, etc.) using Marligen’s proprietary technology. Biotinylated driver RNA and single-stranded (ss)target DNA are made from the custom library and hybridized to each other at a low Cot value (concentration of driver times the time of hybridization).  The resulting hybrids are removed by phenol extraction, the single stranded target DNA is converted to double-stranded DNA and transformed into T1 phage resistant E.  coli. This process reduces the level of abundant and moderately abundant genes and enriches for previously undiscovered rare genes. For normalized libraries, Marligen guarantee a 20-fold reduction in actin compared to the non-normalized library.  However, a 50-100 fold reduction in actin is often observed after normalization. Marligen obtain directionality by using the Not I and EcoRV (blunt end) sites within the pExpress I vector.  However, other restriction sites are also available.

Subtracted Libraries

Subtracted libraries are made from two standard libraries.  One library is used as the driver and one library is the target.  RNA is subtracted from the target library at three different Cot values (concentration of driver times the time of hybridization).  The customer is supplied with both standard libraries plus the three subtracted libraries.

Characteristics of Large Insert cDNA Libraries Constructed from Different Amounts of HeLa mRNA

Comparison of the Size Distribution of Standard and Large-Insert cDNA Libraries prepared by Marligen

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