- Retrieve mini preps for flyPCD assembly from freezer and thaw at room temperature.
- Also let restriction enzymes thaw.
- Do restriction digest (EcoRI/PstI)Diagnostic digest
||15 µl Total
| ||1 rxn||x 9
| DNA plasmid||3||--
| enzyme 1||1||9
| enzyme 2||1||9
| 10x buffer||1.5||13.5
- Aliquot 12 μL to each tube. Then add 3 μL DNA to each tube.
- Place each tube in heat block 37°C.
Follow steps for gel electrophoresis.
- Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
- Fill gel flask with up to 60 ml of TA buffer.
- Create 1% gel by putting .6 grams of agarose into flask.
- Microwave agarose solution for 40 seconds
- Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
- When flask is taken out of microwave, make sure that the agarose is completed dissolved.
- Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
- Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
- Pour gel into tray.
- Wash the agarose gel flask.
- Put tray with gel in electrophoresis appartus.
- Put buffer in apparatus.
- Pipette kb ladder and diagnostic digest samples into wells.
- Turn on the machine and click running man. Set on 100V.
- Check results.
- 1. flyPCD (186) + KAH204 (2328)= 2514
- 2. flyPCD (186) + KAH205 (1251)= 1437
- 3. flyPCD (186) + KAH206 (1551)= 1737
- 4. flyPCD (186) + KAH225 (1251)= 1437
I believe that I made some mistakes when pipetting the DNA into the wells in the gel. For this diagnostic digest, the number of base pairs matching up, but the bands for 1.1 and 1.2 are not there. I'm going to run another diagnostic digest to confirm whether my ligations were successful or not.