WangLab:DNA Amplification
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Make LB plate
- LB broth 12.5 g + Aga power 7.5 g + Water 500 ml
- Autoclave for 1 hr
- Add ampicilin (freezer) 500 ul
Make LB media
- 500 ml water + 12.5 g LB broth
- Autoclave, with no ampicilin
Make EB buffer
- Dilute EB buffer with molecular water for 1x500 times, keep the stock in -20oC.
Day 1 Transformation (Late afternoon, duration: ~2 hours)
- Get the ice bucket and keep DH5a chemical competent cells in ice.
- Turn on the heat shock machine to low setting at 42oC, add water to the surface to make a water bath for even heat-up.
- Add 1 ul DNA to DH5a, keep the vial in ice for 30 min; put 950 ul LB media to round bottom tube (14 ml)
- Heat shock the vial of DH5a for 20 sec at 42oC. If the thermometer reads a higher temperature, add some water to cool it down first.
- Cool down in ice for 2 min
- Add 1 ul DNA mix to LB media, incubate in the warm room for 1 hour.
- Dilute the DNA mix 20 times with LB media: 10 ul DNA mix + 190 ul LB media = 200 ul/dish, add 200 ul DNA mix to the plate
- Soak spreader in ethonal, start gas fire, sterize the spreader using fire 2-3 times, let it cool before spreading DNA.
- Spread the DNA mix with the spreader.
- Put the plate upside down in the incubator for over night (16-18 hours).
- Keep the round bottom tubes for a day in 4oC in case the baterial does not grow well.
Day 2 Amplification
Morning: Check the colonies. If good, seal with parafilm and put in frig to stop growing.
Afternoon:
- Make LB media + Ampicilin (4ml LB media + 4 ul of ampicilin ) and mix, add 4ml mix to each round bottom tube.
- Use 10 ul pipette tip to pick colonies and leave the tips in round bottom tubes.
- Incubate overnight in the warm room for 17-18 hours.
Day 3 Miniprep and Digestion
- Transfer grown mixture to 2 ml tubes
- Centrifuge at 8K RPM for 5 min
If you have 5ml grown mixture in the round bottom tubes, transfer 2 ml mixture at a time and centrifuge for 1.5 min, repeat this step 3 times to get DNA in one tube.
- Use Quagent kit. Add 250 ul of P1 buffer (in frig) to the tube, re-suspend by scratch on the rack (3-4 times).
- Add 250 ul of P2 buffer to the tubes, rock gently with hands (6-8 times), until the fluid turns blue.
- Add 350 ul of N3 buffer to tubes, rock gently (6-10 times). Blue turns white and something can be seen in suspension.
- Centrifuge at 13.2K RPM for 10 min (or 15 min)
- Label the inner tubes. Apply the supernatant to the column (inner tube).
- Centrifuge at 13.2 KRPM for 1 min, discard the liquid.
- Add 750 ul PE buffer/ tube.
- Centrifuge at 13.2K RPM for 1 min
- Discard liquid
- Centrifuge again at 13.2K RPM for 1 min.
- Put the column into a clean vial (with label), elute with 50 ul diluted EB buffer (apply gently).
- Wait for 2 min
- Centrifuge at 13.2K RPM for 1 min.
- Discard column and save the vials with DNA.