Blotting

1. Blotting

2. Blotting • Your nylon filter should now have the DNA from your gel immobilized on the surface • We now want to determine which bands from each restriction digest contain plasmid DNA • To do this, we will hybridize labeled plasmid DNA to your filters, and visualize where it finds complementary plasmid DNA

3. Blotting: Making Labeled DNA linear, denatured plasmid DNA 5’ 3’ random hexanucleotides DNA polymerase (Klenow) dATP, dCTP, dGTP, dTTP digoxigenin-11-dUTP (DIG-dUTP)

4. DIG-dUTP

5. DNA Polymerase Blotting: Making Labeled DNA linear, denatured plasmid DNA 5’ 3’ ATGTTCGTUTGAGUTTGAGGCTA dATP, dCTP, dGTP, dTTP digoxigenin-11-dUTP (DIG-dUTP)

6. AP AP AP AP AP Blotting: Making Labeled DNA ATGTTCGTUTGAGUTTGAGGCTAACGGUGGCGAGCGAUTACGTATGCGTAGG anti-DIG antibody alkaline phosphatase

8. Blotting: Alkaline Phosphate Detection AP enzyme independent X-phosphate (5-bromo-4-chloro-3-indolyl phosphate) clear and soluble 5,5’-dibromo-4,4’-dichloro-indigo blue and insolube reduced NBT purple and insoluble NBT clear and soluble

9. Blotting

10. Blotting: What’s Already Happened • The filters were rinsed briefly in 5X SSC and then incubated in about 20ml of hybridization solution (5X SSC, 0.5% blocking reagent, 0.1% N-laurylsarcosine (sodium salt), 0.02% SDS) for 1-2 hours at 68°C. • The hybridization solution was replaced with 5ml hybridization solution containing 5ng of DIG-labeled denatured plasmid DNA and the incubation was continued at 68°C for 6-24 hours. • Filters were washed, shaking at room temperature, in a large pyrex dish or roller bottles with 2X SSC, 0.1% SDS. This was repeated 5 times with fresh 2X SSC. • Filters were washed at high stringency for 15 minutes at 68°C with 0.1X SSC, 0.1% SDS. This was repeated once.

11. Blotting: Notes on Protocol • DIG/AP labeling replaces radioactivity • There are a lot of washing steps in this protocol. These are important to avoid non-specific background • What is blocking agent for? • The final color reaction is light-sensitive, so do the reaction in a drawer

12. Plasmid Miniprep

13. Miniprep – Qiagen Kit • Step 1: Alkaline lysis of cells • Step 2: Bind plasmid DNA to silica membrane • Step 3: Elute plasmid

14. Miniprep: Alkaline Lysis Start by resuspending cells in a buffer containing: • glucose (to maintain osmotic balance and not burst cells prematurely) • EDTA (to destabilize membranes and inhibit DNAses)

15. Miniprep: Alkaline Lysis Next add NaOH and SDS: • SDS (a detergent) disrupts the cell membrane and creates holes in it • NaOH loosens the cell wall and further breaks down integrity of cell • NaOH denatures DNA - note that, while chromosomal DNA tends to have breaks in it and separates into single strands, while the two strands of plasmid DNA remain together and can renature readily

16. Miniprep: Alkaline Lysis Next add potassium acetate. This: • neutralizes the pH and allows circular DNA to renature • precipitates single-stranded DNA which is insolube in high salt • precipitates the detergent as KDS Spinning the solution removes cell debris and the insoluble ssDNA and KDS

17. Miniprep: Alkaline Lysis The solution now contains circular plasmid DNA and soluble small molecules, and some proteins The plasmid DNA can be recovered by: • precipitation in salt (which neutralizes the DNA) and ethanol/isopropanol • binding to a positively charge substrate (such as the membrane in the Qiagen kit) in low salt (where the DNA is negatively charged) and eluting in high salt