John M. S. Bartlett.pdf - Bio-Nica.info

Subcycling PCR 103
8. Place tubes in thermal cycler and start the program (see Table 1).
9. When cycle is complete, remove PCR products and keep in fridge prior to electrophoresis.
10. Dilute 4 µL of each PCR product into 6 µL of sterile distilled water. This initial dilution
may need adjusting depending on electrophoresis results picture.
11. Electrophorese 1 µL of this diluted DNA on an 0.8% agarose gel.
12. The inversion is associated with a band of 11 kb; the normal allele is associated with a
band of 12 kb. An upper 12-kb band only indicates the inversion is not present; a lower
11-kb band only indicates the inversion is present in an affected male; and bands at both
11 kb and 12 kb indicate a female carrier of the inversion.
References
1. Liu, et al. (1998) Single tube polymerase chain reaction for rapid diagnosis of the inversion
hotspot mutation in haemophilia A (letter). Blood 92, 1458–1459.
2. Liu and Sommer (1998) Sub-cycling PCR for multiplex long distance amplifications of
regions with High and Low GC content: Application to the inversion hotspot in the FVIII
gene. BioTechniques 25, 1022–1028.