I am following this protocol.
Cells from a colony is picked up from agar plates by using a sterile pipette tip and resuspended into 20 μl of of distilled water. Vortex for 10 s for lysing cells. Incubation under 98 °C for 5 min. The lysate was microcentrifuge and the resulting supernatant is collected and subject to qPCR analysis.
This protocol gives me no reproducible results.
Someone has another protocol with consistent results?
Thnks!
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Vortexing will not lyse the cells. In fact Gram positive bacteria are pretty tough with regards to lysis at all. If you want to lyse cells without chemicals try bead beating or sonication. Lately, with Haemophilus at least, I just make a barely turbid (about OD 0.1) suspension in PBS from fresh colonial growth, do a 100 fold dilution of this in PBS, then use 2uL of this as template directly in a 15uL PCR. I suggest you try this, as I have found the boiling method much worse.
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