cDNA Library Construction, cDNA Libraries, Custom cDNA Libraries

We prepare high quality standard, large-insert, normalized, subtracted ,micro-quantity and nano-quantity cDNA libraries using our 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 our Express I vector and transformed into T1 phage resistant E. coli. We 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, we require at least 10 ug of mRNA, 2 mg of total RNA, 2x10⁸ cells or 200 mg of tissue, and we guarantee an average insert size of 3 kb.

Sample Requirements

Library Type	Tissue	Cells	Total RNA	mRNA
Standard cDNA Libraries	≥ 1g	≥ 1x10⁸	≥ 1mg	≥ 5μg
Large Insert cDNA Libraries	≥ 2g	≥ 2x10⁸	≥ 2mg	≥ 10μg
Microquantity cDNA Libraries	≥ 100mg	≥ 1x10⁷	≥ 50μg	≥ 500ng
Nanoquantity cDNA Libraries	≥ 250μg	≥ 25,000	≥ 250ng	≥ 5ng

Guaranteed Results

Library Type	# of primary clones	% Recombinants	Min. average insert size
Standard cDNA Libraries	≥ 3 x 10⁶	≥ 87%	≥ 1 kb
Large Insert cDNA Libraries	≥ 1 x 10⁶	≥ 87%	≥ 3 kb
Microquantity cDNA Libraries	≥ 1 x 10⁶	≥ 87%	≥ 1 kb
Nanoquantity cDNA Libraries	≥ 2 x 10⁵	≥ 87%	≥ 800bp

Normalized Libraries

Normalized cDNA libraries are produced from customers' custom cDNA libraries (i.e., standard libraries, microquantity libraries, etc.) using our 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, we guarantee a 20-fold reduction in actin compared to the non-normalized library. However, we often see 50-100 fold reduction in actin after normalization. We 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.

mRNA (ng)	# of Primary Clones	% Recombinants	Avg. Insert Size (kb)	Insert Size Range (kb)
5000	12 x 10⁷	96	2.24	0.5-4.4
1000	8 x 10⁷	100	2.43	0.9-5.2
500	6.7 x 10⁷	100	1.75	0.8-2.9
100	7.2 x 10⁷	100	1.20	0.5-3.2
5	9 x 10⁵	96	1.01	0.4-3.0
1	4.5 x 10⁴	94	0.92	0.2-3.6

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

mRNA (ng)	# of Primary Clones	% Recombinants	Avg Insert Size (kb)	Insert Size Range (kb)
5000	2.1 x 10⁷	96	4.26	1.0 – 6.2
1000	2.5 x 10⁷	100	4.46	2.4 – 7.2

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

marligen cdna library clone size distribution