1. Southern Hybridization Lab

2. SH SH – done to know whether a specific DNA sequence is present in a DNA sample and where it is located with respect to restriction enzyme sites Restriction digestion + Electrophoresis + Hybridization = contains specific DNA sequence or not

3. Steps Digestion + Gel Agarose gel is soaked in a basic solution – to denature DNA fragments Gel – neutralization buffer Gel is placed on a long piece of blotting paper with ends of paper suspended in a reservoir of salt solution Nitrocellulose or nylon membrane is laid on top of gel

4. Steps Blotting paper and a stack of dry absorbent paper (such as paper towels) are then placed on the membrane Capillary action – fluid is drawn from reservoir up through gel and into stack of dry paper. -Carries denatured DNA fragments up with it out of gel onto membrane (stick forming same pattern) only -Denatured DNA fixed to it through heating or exposure to ultra-violet -Membrane with fixed single stranded DNA – hybridization with desired probe

5. Steps Prehybridization buffer – prevents probe from binding to the membrane in a nonspecific manner Hybridization buffer – contains labeled probe (molecule called biotin) -Solution contains two-component molecule (protein conjugate) -One component is streptavidin which binds tightly to biotin -Other component is enzyme alkaline phosphatase -Both bind to biotin labeled probe.

6. Steps After hybridization – membrane is rinsed repeatedly under conditions that will remove unhybridized and nonspecifically bound probe – this will not disrupt hydrogen bonds between probe and target sequence in sample DNA

7. Color development solution 5-bromo-4-chloro-3 indolyphosphate (BCIP) and nitro tetrazolium (NBT) Alkaline phosphatase portion of conjugate removes a phosphate group from BCIP – resulting product dimerizes to form a dark blue ppt Dimerization step also releases hydride ions that reduce NBT – purple ppt Precipitate is present where ever probe is bound to the membrane – indicating location of DNA fragments hybridized to probe

8. Preparation and experiment schedules April 22 – preparation of solutions April 29 – Steps A, B, C and D April 30 – Steps E, F, and G May 1 – Step H

9. This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal opportunity employer and does not discriminate on the following basis: against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability, political affiliation or belief; and against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United States, or his or her participation in any WIA Title I-financially assisted program or activity.

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