Library quality controls. ( A ) HES3 Human embryonic stem cells were grown and prepared as described ( 9 ). Total RNA was prepared by the Trizol isolation method. A smear of RNA with two bright bands corresponding to the 28S and 18S rRNA was obtained. The ladder used in all panels is Generuler 1 Kb (Fermentas) ( http://www.fermentas.com/catalog/electrophoresis/images/generuler031123.jpg ). ( B ) The mRNA prepared by the use of the μMACS mRNA isolation kit on total RNA showed no bright bands corresponding to the rRNA. ( C ) A flcDNA library was prepared by the Captrapper method, which had a titer of 4.6 × 10 6 cfu. Colony PCR quality control of the library was performed. An empty vector will produce a PCR product of size 260 bp (corresponds to the first band of the ladder); insert sizes were therefore calculated by subtracting off the size of the empty vector. Colony PCR therefore showed a range of insert sizes from 250 to 2000 bp (corresponds to the second to seventh bands of the ladder). This is expected, as a flcDNA library is expected to give a range of different-sized inserts, with no single dominant size. Given that the library was of good quality, as can be seen from the colony PCR, the library was used to prepare two libraries: A single-PET library by the classic method, and a single-PET library by the Selection-MDA method. ( D ) A single-PET library was prepared from the full-length library as per the classic bacterial propagation method. Colony PCR quality control of this library showed a single predominant fixed size of 300 bp in many colonies, which is expected, as single-PET plasmids all have a fixed size of 2800 bp, and hence upon PCR, will give a band of 300 bp. Certain clones do not show this fixed size, which could be the result of the incorporation of foreign DNA, or other factors. Colony PCR quality control showed an insert ratio of 75% based on the number of wells that had PCR products of the correct size (300 bp). 