User:Hetmann/Notebook/2006-11-18

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=Harvard-Yale=

[The Game] Go Harvard! (- and Dartmouth)

Goals for 11/18

 * 1) *Progression
 * 2) ** Step 1: Midiprep
 * 3) **# B0015
 * 4) **# J04500+KaiB
 * 5) **# J04500+KaiC
 * 6) **# J04500+KaiA+J04500+KaiC
 * 7) ** Step 2: Digestion
 * 8) **# B0015/SP important
 * 9) **# J04500+KaiB/XP important
 * 10) **# J04500+KaiC/XP
 * 11) **# J04500+KaiA+J04500+KaiC/SP

Efficient Midiprep Protocol (for HC plasmid)

 * Pre-Midiprep
 * Put buffer P3 in the refrigerator.
 * Check buffer P2 for precipitate; if there is then redissolve @ 37°C.
 * Midiprep
 * Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C.
 * Big centrifuge: SLA-1500; Rotor code 28; 6290 rpm.
 * Big centrifuge: HB6; Rotor code 23; 6060 rpm.
 * Alternatively, centrifuge cultures in 50 mL falcon tubes for 15 minutes at 4400 rmp in room 5096, and dump supernatant. Simultaneously, turn on the big centrifuge in room 5088, fill a bucket of ice, and place the medium rotor into the big centrifuge.
 * Medium rotor: SS34, rotor code 05.
 * Resuspend the bacterial pellet in 4 ml Buffer P1.
 * Add 4 ml Buffer P2, mix by inverting 4-6 times, then incubate at room temperature for 4 minutes.
 * Add 4 ml Buffer P3, mix by inverting 4-6 times, then incubate on ice for 15 minutes.
 * Mix the sample again.
 * Centrifuge at 13,000 rpm for 30 min at 4°C, and remove the supernatant promptly. Concurrently, equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow.
 * Centrifuge in non-glass tube. '<~ this should be all of the tubes present in lab'
 * Centrifuge the supernatant again at 13,000 x g for 5 min at 4°C, and remove the supernatant promptly.
 * Apply supernatant to QIAGEN-tip and allow it to enter the resin by gravity flow.
 * Wash the QIAGEN-tip twice with 10 ml Buffer QC, and allow to move through the QIAGEN-tip by gravity flow.
 * Elute DNA with 5 ml QF.
 * Collect the eluate in a 10 ml tube.
 * Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the eluted DNA.
 * Mix and centrifuge immediately at 11210 rpm for 30 minutes at 4°C, and carefully decant the supernatant. Then make sure all of the isopropanol is out.
 * Wash DNA pellet with 2 ml of room-temperature 70% ethanol and centrifuge at 11210 rpm for 10 minutes.
 * Carefully decant the supernatant, and don't disturb the pellet - nevertheless, make sure that no ethanol remains.
 * Air-dry the pellet for 5-10 min, and redissolve the DNA in 250 ul ddH2O

Estimated time: 170 minutes/ 2h 50m

Adapted from the QIAGEN Midiprep protocol.

Word(s) of the day: karmic retribution