User:Allison K. Alix/Notebook/Thesis Research/2013/03/29

Gel Electrophoresis/FCS

Main project page Previous entry      Next entry

Objectives -Perform Gel Electrophoresis on DNA-AuNP solution as well as supernatants -Perform FCS on MB/DNA-AuNP solutions prepared on 3/26/13 Procedures Part 1: Gel Electrophoresis 1) Dissolve 0.35g agarose in a tupperware container with 35mL 1X TAE buffer Prepare 50mL 1X buffer from 50X buffer 50X(V)=(1X)(50mL) V= 1mL 50X in 49mL H2O 2) Microwave for ~10 seconds at a time for ~1min to dissolve 3) Cool slightly (AKA make sure it isn't boiling and you are able to touch the container safely) 4) Pour into casting tray and allow to solidify (20 to 30 minutes). Make sure the two end pieces and comb are in place. 5) Once solidified, remove the comb, tilting as you pull it out. 6) Prepare the DNA samples including the DNA Ladder Combine 4μL of the following samples with 2μL loading buffer a) 100bp DNA Ladder b) DNA-AuNP Solution c) Supernatant 1 d) Supernatant 2 e) Supernatant 3 f) Supernatant 4 (Note that the ACTUAL protocol for preparing the ladder is 4μL distilled water, 1μL ladder, 1μL loading buffer. This solution was also prepared in addtion to a 4μL water, 1μL buffer, 1μL AuNP-DNA. TOTAL; 8 solutions) 7) Cover the solidified gel with 1X TAE buffer about 1/2cm making sure the wells are full 8) Load each sample into the wells 9) Connect the red and black electrodes (red electrode should be on the side that the DNA will migrate towards) 10) Turn on the voltage source and set to 90V 11) Watch carefully and disconnect once dye is about 3/4 of the way across the gel. 12) Remove from tray and place in tupperware container with 100mL of water and 20μL (10mg/mL) ethidium bromide 13) Stir gently for ~5min to stain 14) Observe under UV light Part 2: FCS 1) Run 3 FCS measurements of the following solutions: 0.2nM MB 0.3nM MB 0.4nM MB 0.5nM MB 0.6nM MB 2) Record <N> and tau