Lactococcal plasmid isolation

Wolfgang Schechinger wolfsc at ibms.sinica.edu.tw
Sat Mar 30 11:49:21 EST 2002


Looks like good old alkaline lysis protocol for E. coli (I think one also can
find it in Sambrook et al.)

> The protocol (DJ O'Sullivan and TR Klaenhammer, 1993) goes this way,
>
> 1. Overnight culture (10 ml) of L. lactis in M17 broth containing 0.5%
> glucose.
> 2. Centrifuge to pellet the cells.
> 3. Discard the supt. and wash pellet with 1 ml of sterile saline.
> 4. Resuspend the pellet in 200 microliters of 25% sucrose containing
> 30 mg/ml lysozyme. Transfer to an eppendorf tube. Incubate at 37°C for
> 15 min.
> 5. Add 400 microliters of alkaline SDS (3% SDS, 0.2N NaOH). Mix and
> incubate at RT/ 7 min.
> 6. Add 300 microliters of ice cold 3M sodium acetate (pH 4.8). Mix
> immediately and spin at 10,000g/ 15 min/ 4°C.
> 7. Transfer supt. to a fresh tube and add 650 microliters of
> isopropanol. Mix well and spin at 10,000g/ 15 min/ 4°C.
> 8. Remove all the liquid and resuspend the pellet in 320 microliters
> of distilled water.
> 9. Add 200 microliters of 7.5M ammonium acetate containing 0.5 mg/ml
> ethidium bromide.
> 10. Add 350 microliters of phenol-chloroform-isoamyl alcohol
> (25:24:1). Mix and spin at 10,000g/ 5 min/ 4°C.
> 11. Transfer aqueous phase to another tube and add 1 ml of absolute
> alcohol. Mix well and spin at 10,000g/ 15 min/ 4°C.
> 12. Wash pellet in 70% ethanol. Air dry the pellet.
> 13. Resuspend the pellet in 40 microliters of TE containing Rnase (0.1
> mg/ml)
> 14. Check 10 microliters in a 0.7% agarose gel.
>
> Papain and Proteinase K were introduced at step 3 or step 8 but no
> effect was observed. Could anyone suggest any modifications or
> alternate protocols?
> Thanks.
>
> Morris
>
>


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Dr. Wolfgang Schechinger
Institute of Biomedical Sciences
Academia Sinica, Taipei, Taiwan

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