HOTSHOT DNA EXTRACTION PROTOCOL

HOTSHOT DNA EXTRACTION PROTOCOL

Modified From Meeker, et al., 2007, Biotechniques vol. 43, 610-614

16 July 2010

1.       Start with a piece of tissue about the size of a grain of rice.

2.       Submerge tissue is 50 mM NaOH. Generally 100 µL is sufficient.

3.       Heat tissue/NaOH mixture on heat block at 95^oC, ideally until tissue is noticeably mushy. This may take anywhere from 10min to 24hrs.

4.       Cool sample to room temperature, and add 1/10 of your starting volume of NaOH of 1M tris at pH=8.0. for example, if you started with 100 µL NaOH, add 10 µL 1 M tris.

5.       Vortex to mix sample.

6.       Centrifuge sample at full speed for 5-10 minutes.

7.       Remove the upper 75% of supernatant  and store in a new tube. This is your DNA extract. Discard tube with pellet and remaining supernatant.

Note : the NaOH/tissue should be vortexed occasionally while heating to help physically break up the tissue into smaller pieces. If desired, you can also cut the “grain of rice” into smaller pieces initially to aid in “dissolving” the tissue. For PCR reactions using DNA extracted with this method, using 1 µL is good starting point. However at times there is excess cellular debris and/or PCR inhibitors remaining in the extract. If initial PCR success is not optimal, a dilution series should be a first troubleshooting step.