User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/07

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] AU CHEM-570 Lab Prep
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Objective

 * Try electroporation
 * Re-ligate with different I:V

Bench work

 * 1) Glycerol stock of ER2566
 * 2) * 250μL 60% sterile glycerol + 750μL ER2566 culture from yesterday &rarr;  80
 * 3) Electrocompetent DH10B
 * 4) * This was carried out with the assistance of User:Daniel Catt
 * 5) * Inoculate 50 mL LB with 1 mL O/N E. coli DH10B culture from yesterday
 * t=0 OD600=0.08
 * t~1.5h OD600~0.2
 * t~2h OD600~0.6 &rarr; stop growth
 * 1) * incubate culture ~10' on ice w/ swirling
 * 2) * spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C | Sorvall RC6+ and SH-3000 rotor
 * 3) ** supernatent
 * 4) *** discard
 * 5) ** pellet
 * 6) *** resuspend in 100 mL cold sterile H2O
 * 7) *** spin in 2x50 mL falcon tube 20' @ 4500 RPM @ 4°C
 * 8) **** supernatent
 * 9) ***** discard
 * 10) **** pellet
 * 11) ***** resuspend in 45 mL cold sterile H2O (should be 50 mL, but not enough sterile water)
 * 12) ***** spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C
 * 13) ****** supernatent
 * 14) ******* discard
 * 15) ****** pellet
 * 16) ******* resuspend in 10 mL cold sterile 10% glycerol
 * 17) ******* spin in 15 mL falcon tube 10' @ 3800 RPM @ 4°C
 * 18) ******** supernatent
 * 19) ********* discard
 * 20) ******** pellet
 * 21) ********* resuspend in 200 μL cold sterile 10% glycerol
 * 22) ********* aliquot into 5 x 50μL (*Kathryn Muratore 15:34, 8 July 2011 (EDT): I meant to freeze the 5th unused aliquot, but did not)
 * 23) Transformation by electroporation
 * 24) * Used David Carlini's Bio-Rad electroporator in Biology
 * 25) * 0.1 cm cuvettes
 * 26) 50μL DH10B + 20μL V(His)+I ligation from [[../../06/30| last week]
 * 27) 50μL DH10B + 20μL V(His) neg ctrl ligation from [[../../06/30| last week]
 * 28) 50μL DH10B + 20μL V+I ligation from [[../../06/30| last week]
 * 29) 50μL DH10B + 20μL V neg ctrl ligation from [[../../06/30| last week]
 * 30) * 1.5 kV, t = 3.6-4.0 ms
 * 31) * add 450μL SOC
 * 32) *&rarr; 1.5h @ 37°C w/ shaking (should be 45')
 * 33) * plate 100μL of 1:1 for all samples on LBAmp100
 * 34) * plate 100μL of 1:100 (diluted with SOC) for V(His)+I and V+I on LBAmp100
 * 35) *&rarr; 37°C O/N
 * 36) Re-plate transformants
 * 37) * spread remaining 450μL of NovaBlue transformations from yesterday
 * 38) *&rarr; 37°C O/N
 * 39) Ligation
 * 40) 8.5μL ddV(His) from  last week + 4μL ddI from  last week + 2μL Ligase buffer + 4.5μL sterile H2O + 1μL Muratore:Materials/DNA ligase
 * 41) 8.5μL ddV from  last week + 4μL ddI from  last week + 2μL Ligase buffer + 4.5μL sterile H2O + 1μL Muratore:Materials/DNA ligase
 * 42) *&rarr; 16°C O/N

Results

 * No transformants on yesterday's plates


 * }