A Laboratory Guide to In Vitro Transcription by Dr. Felipe Sierra (auth.)

By Dr. Felipe Sierra (auth.)

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Add the DTT and Trasylol (but not the PMSF) to this buffer. Due to the viscosity of the solution, this requires vigorously shaking it, until no Schlieren lines are obvious. > Add 10 ml of this buffer to each SW27 tube, to form the pads. Keep these on ice at all times. > Place the Potter-Elvehjem homogenizer in the large plastic beaker with ice-water, and add a few milliliters of homogenization buffer at the bottom of the homogenizer (this is to prevent the tissue from getting stuck at the bottom).

Keep vortexing for about a minute after addition of the nuclei. > From the A26o, calculate the concentration of DNA (1 A260 = 50 Ilglml). 25 mg/ml for spleen and thymus > Add 1/10 volume of 4 M (NH4)2S04, and mix gently by inversion several times. In general, it is better to calculate the amount of (NH4)2S04, mix that with the amount of lysis buffer 48 III Experimental required to dilute the sample (previous step), and then add that mix to the concentrated nuclei. This way there is less possibilities of salting out proteins due to locally high salt concentrations.

After all the salt has dissolved, leave in ice-water, shaking gently, for 20-60 minutes. > Spin for 20 minutes at 35,000 rpm in the Ti 60 rotor (or 40,000 rpm in the Ti 50 rotor). > Aspirate off all the supernatant and discard it. Mark on the tube the position ofthe pellet (it will become very transparent as you start resuspending it). At this point, the pellet can be stored overnight in ice-water. > Resuspend in dialysis buffer by gently pipetting III Experimental 49 up and down, but being careful to avoid foaming.

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