Sequencing

ABI 377 Sequencing - http://people.bu.edu/pbarber/Web%20Protocols/Sequencing%20Cheat%20Sheet.pdf Sequencing using the ABI 377 1. Pour gel. 2. Damp paper towel (~0.5 hr) 3. Wash plates and allow to dry. 4. Put plates in mounting rack. 5. Add bottom buffer tray. 6. Put on plates. 7. Do plate check. 8. Fill bottom buffer tray. 9. Add heating plate. 10. Do pre-run. 11. Prepare samples 12. Load Comb. 13. Fill well with ficoll. - [Read more]

Acrylamide Recipe - http://people.bu.edu/pbarber/Web%20Protocols/Acrylamide.pdf Acrylamide Recipe In a 250 mL beaker add the following: 1 Magnetic Stir Bar 36g Urea (2 vials) 10 mL Long Ranger Gel Solution 20 mL 5X TBE 42 mL DI Water Dissolve Urea in liquids on stir-plate, then filter sterilize. Store at 40C. For Poly-Acrylamide Gels: 1st – Dissolve pre-weighed APS in 500ul ddH2O. This may already be done – do not use APS that is over a week old. - [Read more]

Cycle Sequencing Protocol - http://people.bu.edu/pbarber/Web%20Protocols/Protocol6.pdf Cycle Sequencing Protocol. Cycle sequence is similar to PCR. It uses most of the same ingredients, follows the same basic procedure, and is done in a thermocycler as well. One key difference is that only one primer is used in each cycle sequencing reaction so that the amplification of product is linear, not exponetial. Another key difference is that dideoxynucleotides are used which interrupts the extention of the DNA strand when incorporated. - [Read more]

Membrane Comb Protocols - http://people.bu.edu/pbarber/Web%20Protocols/Membrane%20Comb%20Protocols.pdf Membrane Comb Protocols. Membrane Comb1 Protocols. F. Cipriano, 7/12/2000. Resuspending and Loading Cycle Sequencing Product onto Membrane Combs After you have tried your usual cycle sequencing products, you can determine your own standard conditions for resuspending and loading. For a start, resuspend in 3 ul and load 0.5 ul per tooth. - [Read more]

SEQUENCING PROTOCOL - http://people.bu.edu/pbarber/Web%20Protocols/Sequencing_Protocol-EJones.pdf BARBER LAB SEQUENCING PROTOCOL (ABI 377) Gel Preparation 1. Wash glass plates using 1% Alconox (VWR 21835-032) and a rubber glove. DO NOT let the bottoms of the plates (laser reading area) touch anything hard. 2. Wash Alconox off with warm tap water, then rinse plates well with DD water. While rinsing, rub read area of plate with gloved hands. Rinse with top of plates facing downward so that rinse water moves away from reading area. - [Read more]