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plasmid isolation - Basic Lab Protocols and Techniques

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Old 07-26-2006, 03:11 PM
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Default plasmid isolation



HI
What is difference in procedure and why of plasmid isolation of same size form Gram Negative bacteria like E.coli and Gram positive bacteria like Bacilus??


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Old 07-31-2006, 10:51 PM
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Default Re: plasmid isolation

Gram positive bacteria plasmid isolation is a bit different from gram negative bacteria i believe mainly due to the peptidoglycan layer or "cell wall" present in gram + cells.

From Bio site:
Gram Positive Bacteria Plasmid Isolation Procedure:

Modified protocol of Qiagen and it works with success for me.
For a 250 ml culture of B.subtilis
1) Wash the bacterial pellet once with 100 ml of TSE buffer (10 mM Tris pH8,
300 mM NaCl, 10 mM EDTA) and centrifuge.

2) Resuspend the pellet in 16 ml of P1 containing 5mg/ml lysozyme and RNase
A and incubate at 37°C for 30 min.

3) Add 16 ml of P2 mix by inversion and incubate at room temperature for 5
min.

4) Add 16 ml of chilled P3 mix by inversion incubate on ice 15 min.

5) Centrifuge and follow the classic Qiagen protocol for tip-500.
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