DNA & RNA Isolation - can any person give me protocol for this (Sep/30/2005 )

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There are many protocols for isolating DNA or RNA. gDNA? plasmid DNA?

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gDNA ISOLATION FROM TISSUE CULTURE CELLS WITH PROT. K



Don’t vortex gDNA too rigorously


-Lyse cells or frozen cell pellet in lysis mix, supplemented with prot.K; use 1 ml per confluent 10 cm dish (in 2 ml eppendorf tube)

-Rotate o/n @ 55°C

-Strongly recommended: add same volume of phenol/chloroform/isoamylalcohol, shake well for 30 sec. and spin for 10 min; repeat once or twice when necessary, until interphase has disappeared

- Strongly recommended: after ph/chl/iaa, perform pure chloroform treatment (1:1) to get rid of phenol traces; proceed with upper phase

-Precipitate gDNA by adding equal volume of isopropanol, mix well by inversion (or: use 20 ml mussel glycogen -10 mg/ml stock prepared in mQ- and EtOH precipitate)

-Fish out gDNA with pipettip (or: spin briefly 5K and remove sup)

-Transfer gDNA through eppendorf tube containing 70% ethanol (wash samples separately to avoid contamination)

-Dissolve gDNA in T10E0.1, use 0.4 ml per confluent 10 cm dish (0.1 ml for small pellets); rotate gDNA for 3 hrs to o/n (@37ºC)

-Determine DNA/protein concentration @260/280 nm



Lysis mix (optional: make stock without prot.K for storage @RT)

0.1M TRIS pH 8.5
0.2 M NaCl
5 mM EDTA
0.2% SDS

Add FRESH prot.K to final conc. 100 µg/ml (from 20 mg/ml stock prepared in water, stored @ -20°C)

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RAPID DNA ISOLATION FOR PCR
(also for very small amounts of cells)



- Take cells from a 60 mm dish or less, or about 50 mg of (embryonal) tissue

- Use 4x pellet volume to resuspend in 30 ml - 500 ml PKB

- Try to make cell suspension (to allow prot. K to work more efficiently)

- Incubate while vortexing/shaking (e.g. Eppendorf incubator) @ 55˚C for at least 3-5 hrs, for large quantities o/n

- Heat up to 80-90 °C (boil) for 10 min (to kill prot. K)

- Spin max for 2 min @ 4°C to pellet crude stuff

- Put sup into new tube on ice

- EITHER: Use 1 µl for 20 µl PCR reaction; store remaining solution @ -20°C

- OR (preferably): First perform phenol (1 or 2x) treatment; for very small amts: use siliconized eppies

- Precipitate with equal volume of isopropanol:
- either fish out DNA (that will make RNAse treatment unnecessary)
- or spin 10 min max @ 4°C

- Wash with 70% EtOH

- Carefully dissolve DNA @ 37 °C in TE (100 ml per 10 cm plate)

- When necessary: RNAse treatment, followed by second phenol treatment and precipitation, wash

- Determine DNA concentration

- Use at least 100 ng DNA for PCR





PKB:

0.1 mg/ml gelatin
10 mM TRIS pH 8
50 mM KCl
2.5 mM MgCl2
0.5% Tween 80 (20)
0.1 mg/ml prot. K (20 mg/ml stock - aliquot stock!)

Aliquot prot. K-containing solutions and use once - no freeze/thaw

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hi
this forum is a part of www.protocol-online.org which allows you to find several answers...
fred

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