20.109(F12): TA notes for module 2: Difference between revisions
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*Streak out NB466 (= bacterial photography strain, alternative = NB334) on LB+Cam34+Amp25+Kan10 | *Streak out NB466 (= bacterial photography strain, alternative = NB334) on LB+Cam34+Amp25+Kan10 | ||
*Streak out NB467 and NB468 (= kinase dead strain H557A, in XL1-blue and photography strain respectively). NB467 should be streaked out on LB+Cam34 and NB468 on LB+Amp25+Cam34+Kan10. If there is some problem with this "kinase dead" pair of strains, then it's also possible to use I566V mutant, NB469 and NB470. | *Streak out NB467 and NB468 (= kinase dead strain H557A, in XL1-blue and photography strain respectively). NB467 should be streaked out on LB+Cam34 and NB468 on LB+Amp25+Cam34+Kan10. If there is some problem with this "kinase dead" pair of strains, then it's also possible to use I566V mutant, NB469 and NB470. | ||
*'''prepare electroporation competent cells of NB462.''' Innoculate 3x 50 ml LB+Amp25+Kan10 with 50 ul of an overnight culture of NB462 that was grown in LB+Amp25+Kan10. For F12: grown in 3x125 ml flasks in room temp shaker starting at 4PM and harvested at 3PM next day. Pool cultures in one flask to make uniform (measured OD600 = 0.408, should be higher!). Divide between 4x50 ml conical tubes of ~35 ml each to spin in centrifuge in swinging bucket rotor in cold room 5K rpm 5'. Resuspend cells from each 50 ml conical in 10 ml cold 10% glycerol (40 ml total) and spun again. Repeat 10 ml wash. Resuspend cells in each tube with 1 ml cold 10% glycerol. Move to 20 eppendorfs with | *'''prepare electroporation competent cells of NB462.''' Innoculate 3x 50 ml LB+Amp25+Kan10 with 50 ul of an overnight culture of NB462 that was grown in LB+Amp25+Kan10. For F12: grown in 3x125 ml flasks in room temp shaker starting at 4PM and harvested at 3PM next day. Pool cultures in one flask to make uniform (measured OD600 = 0.408, should be higher!). Divide between 4x50 ml conical tubes of ~35 ml each to spin in centrifuge in swinging bucket rotor in cold room 5K rpm 5'. Resuspend cells from each 50 ml conical in 10 ml cold 10% glycerol (40 ml total) and spun again. Repeat 10 ml wash. Resuspend cells in each tube with 1 ml cold 10% glycerol. Move to 20 eppendorfs with 200 ul each. Pellet. Resuspend cells in 50 ul cold 10% glycerol for a total of 20 epps of EP comp tubes and freeze -80° <6 months. '''Test electroporation before module starts.''' | ||
*miniprep H557A kinase dead control plasmid from NB467 and/or I566V mutant from NB469. These will be used for electroporation/screening controls on Day 3. | *miniprep H557A kinase dead control plasmid from NB467 and/or I566V mutant from NB469. These will be used for electroporation/screening controls on Day 3. | ||
Revision as of 12:49, 8 August 2012
Before module begins
Fall 2012 version
Archive
Fall 2011
Archive of TA notes
Fall 2009
Fall 2010,
Strains
- Streak out NB5 (= b-gal overproducing strain) on LB+Amp100
- Streak out NB462 (= strain for transformation/library screen) on LB+Amp25 (can also spread plate with 25 ul Kan10 to check strain)
- Streak out NB466 (= bacterial photography strain, alternative = NB334) on LB+Cam34+Amp25+Kan10
- Streak out NB467 and NB468 (= kinase dead strain H557A, in XL1-blue and photography strain respectively). NB467 should be streaked out on LB+Cam34 and NB468 on LB+Amp25+Cam34+Kan10. If there is some problem with this "kinase dead" pair of strains, then it's also possible to use I566V mutant, NB469 and NB470.
- prepare electroporation competent cells of NB462. Innoculate 3x 50 ml LB+Amp25+Kan10 with 50 ul of an overnight culture of NB462 that was grown in LB+Amp25+Kan10. For F12: grown in 3x125 ml flasks in room temp shaker starting at 4PM and harvested at 3PM next day. Pool cultures in one flask to make uniform (measured OD600 = 0.408, should be higher!). Divide between 4x50 ml conical tubes of ~35 ml each to spin in centrifuge in swinging bucket rotor in cold room 5K rpm 5'. Resuspend cells from each 50 ml conical in 10 ml cold 10% glycerol (40 ml total) and spun again. Repeat 10 ml wash. Resuspend cells in each tube with 1 ml cold 10% glycerol. Move to 20 eppendorfs with 200 ul each. Pellet. Resuspend cells in 50 ul cold 10% glycerol for a total of 20 epps of EP comp tubes and freeze -80° <6 months. Test electroporation before module starts.
- miniprep H557A kinase dead control plasmid from NB467 and/or I566V mutant from NB469. These will be used for electroporation/screening controls on Day 3.
