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HotStarTaq PCR Handbook

orage of single cells: Single cells may be isolated by different methods (e.g., by flow cytometry or by micromanipulation).
Keep samples cool during the cell-isolation procedure to prevent DNA degradation.
Transfer cell into a PCR tube that has been filled with 20 l of 1x PCR Buffer. Immediately freeze the sample on dry ice.
Store cell at 80C until required for PCR analysis. PCR setup: Prepare a fresh master mix for single-cell PCR (see below).
Thaw the cells on ice.
Distribute 30 l of the master mix into each PCR tube, and place the tubes in the thermal cycler. Immediately start the cycling program with a 10 min incubation step at 95C to activate HotStarTaq DNA Polymerase for single-cell PCR. 50 cycles of PCR may be required to amplify a single-copy gene in one round of PCR. Master mix preparation: Prepare a master mix that has a final volume of 30 l per PCR, as detailed below.
Notes:
Addition of carrier nucleic acid is usually required (e.g., E. coli 5S rRNA).
Use polyacrylamide gel- or HPLC-purified primers only. Component Volume/reaction Final concentration
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