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MOLECULAR BIOLOGY: WORKING WITH DNA

ENZYMATIC MANIPULATION

RAPID BIDIRECTIONAL ALKALINE SOUTHERN BLOT FOR CLONED DNA

Rapid Bidirectional Alkaline Southern Blot for Cloned DNA Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	The Laboratory of Michael Koelle at Yale University. URL: Lab Web Site
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
The procedure uses Nytran paper for southern transfer of DNA from an agarose gel. The procedure is specific to Nytran paper; nitrocellulose paper will not work. Large DNA fragments (greater than approximately 5 kb) will not transfer very well.
Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
A. Gel Processing 1. Electrophoresis the DNA sample in a 0.5% to 1.2% Agarose Gel and include Ethidium Bromide (see protocol on Gel Electrophoresis of DNA). 2. Expose gel to UV light for 1 min on 265 nm light box (CAUTION, see Hint #1, for an alternate method, see Hint #2). 3. Wash gel with 2 volumes of Hydroxide/Chloride Solution for 15 min on a shaking platform (see Hint #3). 4. Decant the Hydroxide/Chloride Solution. 5. Wash gel with 2 volumes of Hydroxide/Chloride Solution for 15 min on a shaking platform. 6. Discard the Hydroxide/Chloride Solution. B. DNA Transfer 1. Cut 2 sheets of Nytran and 6 sheets of 3MM Filter Paper to the approximate size of the gel. 2. Saturate the sheets in Hydroxide/Chloride Solution. 3. Lay down a 2 inch layer of paper towels. 4. Lay 3 sheets of saturated 3MM Filter Paper down on the paper towel and lay 1 sheet of saturated Nytran on top of the 3MM Filter Paper. 3. Lay the gel on top of the saturated Nytran sheet. 4. On the exposed side of the gel, lay 1 sheet saturated Nytran. 5. On top of the saturated Nytran sheet lay 3 sheets of saturated 3MM Filter Paper. 6. Lay down another 2 inch layer of paper towels on top of the 3MM Filter Paper and gel stack. 7. Apply even weight on top of the gel sandwich to ensure even contact of the gel with the two sheets of Nytran. 8. Allow transfer to occur at room temperature for at least 1 hour (continue to apply the even pressure). 9. Remove the Nytran paper from the gel sandwich and rinse for 2 min in 2X SSPE Solution. 10. UV crosslink DNA to the Nytran paper in a Stratalinker (for an alternative method, see Hint #4).
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Hydroxide/Chloride Solution    1.5 M NaCl 0.5 M NaOH 0.25 M HCl SSPE (2X)    add ddH2O to 900 ml 175.3 g NaCl pH to 7.4 7.4 g EDTA 27.6 g Sodium Phosphate Monobasic (NaH2PO4) add ddH2O to 1 liter
BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Sodium Hydroxide Hydrochloric Acid Agarose Sodium Phosphate, Monobasic EDTA Sodium Chloride
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. Never look at a UV light source without proper eye protection. 2. Alternatively you can depurinate in 2 volumes of 0.25 M Hydrochloric Acid 2 times for 15 min each time. Then rinse thoroughly in ddH2O. 3. This is to break and denature the DNA inside the gel. 4. Bake the Nytran paper at 80°C in a vacuum oven for 30 min to 2 hours.