I have to obtain bacterias naked without peptoglycan layer, also trying to not kill the cell. Thanks in advance.
Can you tell us the species of the bacteria and what you know about what osmolalities it likes? As I understand it, stripping of the peptidoglycan with lysozyme should be relativity easy, stopping the cell exploding will be much harder!
A good place to start could be using the same buffers that are used to keep competent cells alive such as 0.1M CaCl.
1. Suspend the cell pellet of the (up to 1× 106 cells)or the pellet of 2 ml broth culture in 200 ul of 1X TE buffer.
2. Add 20 ul of lysozyme (50mg/ml) and incubate the tube OVERNIGHT at 37 for at (YOU must now that 1 hr incubation is NOT ENOUGH)
3. Centrifuge the tube at 13000 rpm for 5 min. and discard the supernatant
4. Add 200 μl of deionized water and mix well
5. Add 100 μl of Isopropanol and mix well by pippetting. Don’t vortex as this might reduce DNA yield.
6. Carefully transfer it into the upper reservoir of the BINDING COLUMN TUBE fited in a tube.
7. Close the tube and centrifuge at 8,000 rpm for 1 min. If the lysate has not completely passed through the column after centrifugation, centrifuge again at a higher speed (>10,000 rpm) until the binding column tube is empty.
8. Open the tube and transfer the Binding column tube to a new tube for filtration.
9. Add 500 ìl of Washing buffer without wetting the
rim, close the tube, and centrifuge at 8,000 rpm for 1 min.
10. Transfer the Binding column tube to a new 1.5 ml tube for elution (supplied), add 200 μl of Elution buffer (EL, or nuclease-free water) onto Binding column tube, and wait for at least 1 min at RT (15~25℃) until EL is completely absorbed into the glass fiber of Binding column tube.
11. Centrifuge at 8,000 rpm for 1 min to elute. About 180 μl ~ 200 μl of eluent can be obtained when using 200 μl of Elution buffer (or nuclease-free water).
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