I grow my positive clones in liquid LB (100 ul) for 3-4 hours until the solution is turbid enough. I want to know how much of the template is sufficient to put in a colony PCR reaction since the concentration of plasmid or DNA template is unknown!
What I usually do and works just fine is the following:
- Pick a single colony from your selection plate
- Dilute it in 12μl nuclase-free water (the one you use for your PCR)
- Use 2μl from the above as a template for a 20μl reaction
- If the colony is positive, grow the remaining 10μl on selective media.
Good luck.
With colony PCR or PCR from a culture the problem is often too much template and never too little. With colony PCR I pick a little bit of colony with pipet tip and put in 100ul water, boil and then use 1ul as template. With culture I spin down about 100ul of cells, resuspend in 100ul of water, boil and use 1ul.
I use an approach like yours (colony in 100 ul with selection- this is key!) but only shake about 1 hour. I use 2 ul as my template for a 20 ul PCR reaction.
Be sure to use selective media for the tiny culture to keep selective pressure or your cells can lose the plasmid.
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