Shreffler:Notebook/retinoic acid trans signaling/2009/05/21

Retinoic Acid Trans Signaling

Prepare 1% Agarose Gel:

(Note: Formula for 40 mls)

1. Take out 0.4g of LE Agarose powder, and mix with 40 ml of TAE buffer

2. Add 1 ul of ethidium bromide

3. Heat in microwave for 30 sec to 1 min

4. Allow to cool (so there are no bubbles), and place in the tray to dry

5. Run between 80 to 100 Volts for approx 45 min to 1 hour

Calculations for Agarose Gel: (Want a concentration of 250 ng/ul)

Loading Buffer = 5x concentration

1x sample: 189.7 ng/ul = 250/189.7 = 1.3 ul of DNA extract

1.3 ul of DNA extract + 2 ul of loading buffer + 6.7 ul of H2O = 10 ul total

2x sample: 112.3 ng/ul = 250/112.3 = 2.2 ul of DNA extract

2.2 ul of DNA extract + 2 ul of loading buffer + 5.8 ul of H2O = 10 ul total

Plasmid sample: 500 ng/ul = 250/500 = 0.5 ul of DNA extract

0.5 ul of DNA extract + 2 ul of loading buffer + 7.5 ul of H2O = 10 ul total

Agarose Gel Layout:

1st well: 1 Kb Plus Ladder 250 ug (1.0 ul/ul) = 10 ul

3rd well: 10 ul of 1x sample

5th well: 10 ul of 2x sample

7th well: Plasmid sample

Results of agarose gel: Used to compare purified plasmid from yesterday's preps to material Ananth gave us:

Media:gel_052209

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