PCR product too short
Posted 29 November 2010 - 01:53 PM
Posted 29 November 2010 - 02:35 PM
Posted 29 November 2010 - 02:38 PM
Posted 29 November 2010 - 04:35 PM
Posted 30 November 2010 - 02:18 PM
To set up the PCR properly you should be doing annealing temperature and magnesium gradients to determine the optimal conditions. You should also only use a tiny anount of DNA for plasmid based reactions. 10 ng is heaps.
Posted 01 December 2010 - 06:30 AM
Adding DMSO at 8% of the final volume of rxn helped produce strong bands at the desired size for me, without DMSO i was getting non specific binding everywhere and nothing at the size i was after. And wish i had found out about it before trying to tweak all the salt concentrations/dNTPs etc.
Hope this helps. (Wear gloves as im told DMSO can burn on contact with skin)
Edited by Chris22, 01 December 2010 - 06:32 AM.
Posted 02 December 2010 - 02:20 PM