Primary PCR: The PCR reactions are performed using tagged genome specific primers in the presence of genomic DNA (provided by your laboratory). We normally achieve a success rate of 93 % of PCR reactions. For the failed PCR reactions we perform three different protocols of PCR conditions, which include different MgCl2 concentrations and different Taq polymerase. We should normally get an additional 2 % working and reach a success rate of 95 % in total. We check 5 % of the total oligos by Mass Spectrometry and will re-synthesize maximum 2 % of the oligos based on a newly designed primer. We will perform the PCR reactions with the new designed oligonucleotides with the standard PCR protocol. Following this process we reach usually 97 % success rate. Secondary PCR: amplifications will be performed using a single pair of primers annealing to tag sequences introduced on primary PCR primers. These PCR amplifications will also be controlled by agarose gel electrophoresis and repeated if required reaching a minimum of 95 % success rate. The final PCR products will contain an amino group at their 5' end to promote the attachment of the predicted gene coding DNA strand to the treated glass slides.
Support
glass
Coating
unknown
Description
22176 IGRs and ORFs were spotted in duplicate on a glass slide
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