Spot synthesis manual

Spot synthesis may be carried out manually, semi- or fully automated (see Fig. 1). Manual spot synthesis is convenient for the synthesis and screening of a relative small number of peptides (up to 100) and a large pipetting volume (>0.5 aL). For all other cases a semiautomated or fully automated synthesis is recommended. For synthesis of small spots with a pipetting volume of about 0.1 ptL, spot synthesizers from Intavis AG (Koln, Germany) are very useful (3). 

SPOT-synthcsis is particularly flexible with respect to numbers and scales that can be accomplished. The arrays arc freely selectable to fit the individual needs of the experiment by the choice of membrane material, thickness, area specific anchor loading, and spot size Table 1). The standard format used in manual SPOT-synthesis was adapted to the 8 X 12 array of a microtitre plate with 96 spots Figure, ). 

A kit that includes all necassary items for manual SPOT-synthesis is currentiy avaii-able from GENOSYS... 

Fmoc-(3-alanine/DMSO solution to all spot positions (see Note 4). For manual synthesis we recommend marking the spot positions with a pencil and to use a multistep pipettor (see Fig. 2). Repeat the delivery after 20 min. After that allow the reaction to occur once more for at least 20 min. 

A list of peptides to be synthesized was prepared and the number of peptides in the required array, the spot size, and the scale were defined. The spot positions were marked on the membrane with pencil dots for manual synthesis. The membrane sheet was placed in the reaction trough. An NMP solution, which was 0.3 M with respect to the second anchor compound [e.g., Fmoc-PAla or Boc-Lys(Fmoc)], 0.45 M with respect to HOBt, and 0.35 M with respect to DIC, was prepared and allowed to stand for 30 min. Aliquots of this solution were then spotted onto all positions according to the chosen array configuration. After 15 min, the spotting was repeated and reactions were allowed to proceed for 30 min. The sheet was washed with 2% AC2O/DMF (20 mL) for 30 s, once again for 2 min, and finally left in the solution overnight. The array was washed with DMF (3 x 20 mL), incubated for 10 min with 20% pi-... 

Mark the spot positions on the membranes with pencil dots for manual synthesis and place the membranes in the reaction trough or fix them on the platform of the ASP222-robot. ... 

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