Recently, we developed methods that enable us to produce High Quality Nanoquantity cDNA Libraries from such small amounts of tissue and cells that material prepared by laser capture microdissection (LCM) or FACS can be used. Like our standard libraries, these libraries are directionally cloned into Express I and then transformed into T1 phage resistant E. coli. We guarantee at least 2×105 primary clones with ≥87% recombinant clones containing an average insert size of at least 800 bp, however, we generally produce libraries of 5×105 primary clones with >95% recombinant clones containing ≥1.0kb average insert size.
We will make nanoquantity libraries from customer supplied tissue (at least 250 µg), cells (at least 25,000), total RNA (≥250 ng) or mRNA (≥5 ng). We provide quality controlled primary or amplified libraries.
Characteristics of a cDNA Library Constructed From a Nanoquantity Amount of HeLa mRNA
|mRNA (ng)||Total # of primary clones||Average insert size (kb)*||Percent recombinants||Insert size range (kb)*|
|100||7.2×107||1.20||100||0.5 – 3.2|
|50||1.5×107||1.35||100||0.6 – 3.5|
|10||1.7×106||1.00||100||0.4 – 2.9|
|5||9.0×105||1.01||96||0.4 – 3.0|
|2||2.0×105||0.74||94||0.2 – 2.3|
|1||4.5×104||0.92||94||0.2 – 3.6|
mRNA (ng), Total # of primary clones, Average insert size (kb)*, Percent recombinants, Insert size range (kb)**Average insert size and insert size range determined by restriction enzyme digestion of 24 clones picked at random from each library.
Quality testing: Percentage of recombinant clones and average insert size determined by gel analysis of 24 clones picked at random.
Shipment and storage conditions: Libraries will be shipped in dry ice. Storage of libraries at -80°C is recommended.