User:Jehane Ibrahim Eid/Notebook/Molecular Biology/2010/08/08

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HotSHOT* genomic DNA preparation

 * HotSHOT* genomic DNA preparation

hot sodium hydroxide and tris

from Biotechniques. 2000 Jul;29(1):52,54

obtained from the Camper Lab

Notes before starting:

· DNA is suitable for PCR reactions

o TOO MUCH TISSUE WILL NOT WORK FOR PCR.

o Use 0.5-5 μl of the reaction product for PCR.

· DNA is NOT suitable for Southerns

o This is because the protocol yields relatively short DNA segments

· Heating for longer than 30 minutes does not increase DNA concentration

o Accidentally heating for an entire weekend does not negatively affect DNA concentration

· Do not worry about undigested floating tissue – tail snips often won’t look like anything has happened to them, but the DNA is still there.

· DNA should be stored at 4ºC or -20ºC.

o If you are taking tail snips, a good amount is about this size: v

Protocol:

1. Obtain tissue and place in a tube. 1. If you are taking tail snips, a good amount is about this size: v         2. Use a .65 mL tube if you plan on heating in the thermocycler 3. Can use a 1.7 mL tube if you plan on heating in the sand block. 2. Add 75 μl Alkaline Lysis Reagent. 3. Heat sample to 95ºC for 10 minutes to an hour (30 minutes is optimal) 4. Cool to 4ºC (optional). 5. Add 75 μl Neutralization Buffer. 6. DNA can be used immediately.

Buffers:

Alkaline Lysis Reagent

NaOH	Final Conc. 25 mM   Amount for 200 mL    200 mg

EDTA   Final Conc. 0.2 mM   Amount for 200 mL    14.88 mg

Neutralization Buffer

Tris-HCl	Final Conc. 40 mM   Amount for 200 mL    1.3 g

Add ddH2O to a final volume of 200 mL. pH of Alkaline Lysis Reagent will be 12. pH of Neutralization Buffer will be 5. There is no need to pH these solutions.


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