PCR

5X TBE Buffer - http://people.bu.edu/pbarber/Web%20Protocols/Protocol7.pdf 5X TBE(1.1M Tris; 900mM Borate; 25mM EDTA; pH 8.3) REAGENT 8L 2L Sigma Aldrich Cat# disodium EDTA 37.5g 9.375g E-5134 Trizma Base 432g 108 g T-1503 Boric Acid 220g 55g B-0252 In an appropriately sized beaker, dissolve Tris, Boric Acid and EDTA in a volue of distilled deionized water that is equal to about 75% of the volume of 5X buffer to be made. - [Read more]

Agarose Electrophoresis - http://people.bu.edu/pbarber/Web%20Protocols/Protocol4.pdf Agarose Electrophoresis. E. Crandall/P.Barber. Agarose elctrophoresis is performed to visualize your PCR products. This step allows you to determine whether your PCR was successful, whether the resulting product is the correct size, whether other products were amplified as well, and whether the concentration of the resulting product is suitable for cycle sequencing. All gel work should be performed in the HIGH DNA area. - [Read more]

Double-Stranded Amplification PCR - http://people.bu.edu/pbarber/Web%20Protocols/Protocol3.pdf PCR (For Hot-Start, Double-Stranded Amplifications). PCR will make about 40 Billion copies from a given template. Because of this, it is imperative to maintain excellent sterile technique throughout as any contaminant will be amplified along with (or instead of) your template DNA. Work in the LOW DNA portion of the lab. - [Read more]

Gel documentation system protocol - http://people.bu.edu/pbarber/Web%20Protocols/Gel%20Documentation%20System.pdf Gel Doc System 1. Turn camera on. To access camera lift the top cover of the hood. 2. Open Kodak Imaging program by clicking on desktop icon. 3. Click on Capture Image button on top left of screen. 4. Place stained gel on UV box. Position gel on left edge and center. 5. Close hood door. Turn on UV. 6. Click on Take Picture button. 7. After photo appears on screen turn offf UV. - [Read more]

PCR CLEAN-UP - http://people.bu.edu/pbarber/Web%20Protocols/SAP%20Exo%20PCR%20Clean%20Up-SLB%20.pdf PCR CLEAN-UP. SAP/EXO1 PCR CLEAN-UP (chew up excess primers, remove dNTPs) 1. Visualize PCR product to make sure that you have one without sub-bands. A strong amplification product (bright on the gel) will tend to sequence better. Concentration should be at least 20ng/μL as visualized using the MassLadder 2. Add 5 μL of each PCR product into a 200μL strip tube: - [Read more]

PCR product clean-up protocol - http://people.bu.edu/pbarber/Web%20Protocols/Protocol5.pdf PCR product “clean-up” protocol: SAP/EXO The purpose of this procedure is used to chew up excess primers and remove excess dNTPs from your PCR product. This proceedure is necessary to ensure clean and readable DNA sequences. Work in HIGH DNA part of lab. - [Read more]