Bacterial stock plates

The source material was diluted 1:10 in sterile Milli-Q water

Primers (Sigma Genosys)

Ko forward primer (amidated): GTG TGG AAT TGT GAG CGG ATA ACA A (25mer) with 5' NH₂(C₆) modification.
Ko reverse primer: CCA GTC ACG ACG TTG TAA AAC GAC (24mer)

PCR amplification

PCR was performed in Abgene 96-well PCR thermofast plates on a KBiosystems dunking PCR Thermal Cycler, using Hotstar Taq (Qiagen) and dNTPs (Amersham Pharmacia Biotech) with the following cycling parameters:

Purification

The PCR products were purified using Millipore multiscreen PCR plates resulting in a 3-fold concentration in sterile MilliQ water.
To confirm the integrity of the DNA, purified PCR products were run on agarose gels, together with marker bands (New England Biolabs 1 kb DNA ladder) and visualised using a Syngene Gel Analysis system. These quality checks showed that 95% of the wells produced PCR products with 94% of the products comprising a single band. These gel images can be viewed here.

Aliquots

10µl aliquots were dispensed into Abgene 96-well thermofast plates using a Matrix PlateMate Plus robot and flash-frozen on dry ice after plate sealing with Abgene EasyPeel plate sealers. Plates were immediately stored at -20°C after the initial flash freeze. QC has been performed in-house to establish that there is no degradation in product when stored at this temperature.

Storage

We recommend that the plates containing the PCR products are stored at -20°C.

Concentration

The average concentration is 0.1µg/µl (determined by A₂₆₀ absorbance).
10µl PCR product was dispensed into each well. There may have been some evaporation of water during storage, despite the plate sealer. However no PCR product will have been lost. If evaporation appears to have occurred the wells can be topped up with sterile MilliQ water. The PCR products have been supplied in water to allow for the addition of appropriate buffers prior to spotting on glass microarray slides.
The in-house HGMP microarraying group add 20µl of spotting buffer to the 10µl of PCR product and have found that this is sufficient to grid over 1000 glass arrays.

Acknowledgements

Please acknowledge the originators of this mouse cDNA clone set in any publications arising out of this work:
Tanaka TS, et al Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray. PNAS (2000) 97: 9127-32

Version 1.1 28^th Nov. 2001