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

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#* This was carried out with the assistance of [[User:Daniel Catt]]
#* This was carried out with the assistance of [[User:Daniel Catt]]
#* Inoculate 50 mL LB with 1 mL O/N ''E. coli'' [[DH10B]] culture from [[../06#Bench work| yesterday]]
#* Inoculate 50 mL LB with 1 mL O/N ''E. coli'' [[DH10B]] culture from [[../06#Bench work| yesterday]]
## t=0 OD<sub>600</sub>=0.08
#: t=0 OD<sub>600</sub>=0.08
## t~1.5h OD<sub>600</sub>~0.2
#: t~1.5h OD<sub>600</sub>~0.2
## t~2h OD<sub>600</sub>~0.6 &rarr; stop growth
#: t~2h OD<sub>600</sub>~0.6 &rarr; stop growth
#* incubate culture ~10' on ice w/ swirling
#* incubate culture ~10' on ice w/ swirling
 
#* spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C [http://www.thermoscientific.com/ecomm/servlet/productsdetail_11152_L10536_88614_12774454_-1| Sorvall RC6+ and SH-3000 rotor]
#** supernatent
#*** discard
#** pellet
#*** resuspend in 100 mL cold sterile H<sub>2</sub>O
#*** spin in 2x50 mL falcon tube 20' @ 4500 RPM @ 4°C
#**** supernatent
#***** discard
#**** pellet
#***** resuspend in 45 mL cold sterile H<sub>2</sub>O (should be 50 mL, but not enough sterile water)
#***** spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C
#****** supernatent
#******* discard
#****** pellet
#******* resuspend in 10 mL cold sterile 10% glycerol
#******* spin in 15 mL falcon tube 10' @ 3800 RPM @ 4°C
#******** supernatent
#********* discard
#******** pellet
#********* resuspend in 200 μL cold sterile 10% glycerol
#********* aliquot into 5 x 50μL (*'''[[User:Kathryn Muratore|Kathryn Muratore]] 15:34, 8 July 2011 (EDT)''': I meant to freeze the 5th unused aliquot, but did not)
# Transformation by electroporation
#* Used David Carlini's Bio-Rad electroporator in Biology
#* 0.1 cm cuvettes
## 50μL DH10B + 20μL V(His)+I ligation from [[../../06/30| last week]
## 50μL DH10B + 20μL V(His) neg ctrl ligation from [[../../06/30| last week]
## 50μL DH10B + 20μL V+I ligation from [[../../06/30| last week]
## 50μL DH10B + 20μL V neg ctrl ligation from [[../../06/30| last week]
##* 1.5 kV, t = 3.6-4.0 ms
##* add 450μL [[SOC]]
##*&rarr; 1.5h @ 37°C w/ shaking (should be 45')
##* plate 100μL of 1:1 for all samples on LBAmp<sup>100</sup>
##* plate 100μL of 1:100 (diluted with SOC) for V(His)+I and V+I on LBAmp<sup>100</sup>
##*&rarr; 37°C O/N
# Re-plate transformants
#* spread remaining 450μL of NovaBlue transformations from [[../06| yesterday]]
##*&rarr; 37°C O/N
# Ligation
## 8.5μL ddV(His) from [[../../06/29| last week]] + 4μL ddI from [[../../06/29| last week]] + 2μL Ligase buffer + 4.5μL sterile H<sub>2</sub>O + 1μL [[Muratore:Materials/DNA ligase]]
## 8.5μL ddV from [[../../06/29| last week]] + 4μL ddI from [[../../06/29| last week]] + 2μL Ligase buffer + 4.5μL sterile H<sub>2</sub>O + 1μL [[Muratore:Materials/DNA ligase]]
##*&rarr; 16°C O/N


==Results==
==Results==

Revision as of 12:34, 8 July 2011

AU CHEM-570 Lab Prep <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Objective

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

Bench work

  1. Electrocompetent DH10B
    t=0 OD600=0.08
    t~1.5h OD600~0.2
    t~2h OD600~0.6 → stop growth
    • incubate culture ~10' on ice w/ swirling
    • spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C Sorvall RC6+ and SH-3000 rotor
      • supernatent
        • discard
      • pellet
        • resuspend in 100 mL cold sterile H2O
        • spin in 2x50 mL falcon tube 20' @ 4500 RPM @ 4°C
          • supernatent
            • discard
          • pellet
            • resuspend in 45 mL cold sterile H2O (should be 50 mL, but not enough sterile water)
            • spin in 50 mL falcon tube 20' @ 4500 RPM @ 4°C
              • supernatent
                • discard
              • pellet
                • resuspend in 10 mL cold sterile 10% glycerol
                • spin in 15 mL falcon tube 10' @ 3800 RPM @ 4°C
                  • supernatent
                    • discard
                  • pellet
                    • resuspend in 200 μL cold sterile 10% glycerol
                    • aliquot into 5 x 50μL (*Kathryn Muratore 15:34, 8 July 2011 (EDT): I meant to freeze the 5th unused aliquot, but did not)
  2. Transformation by electroporation
    • Used David Carlini's Bio-Rad electroporator in Biology
    • 0.1 cm cuvettes
    1. 50μL DH10B + 20μL V(His)+I ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
    2. 50μL DH10B + 20μL V(His) neg ctrl ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
    3. 50μL DH10B + 20μL V+I ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
    4. 50μL DH10B + 20μL V neg ctrl ligation from [[User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/30| last week]
      • 1.5 kV, t = 3.6-4.0 ms
      • add 450μL SOC
      • → 1.5h @ 37°C w/ shaking (should be 45')
      • plate 100μL of 1:1 for all samples on LBAmp100
      • plate 100μL of 1:100 (diluted with SOC) for V(His)+I and V+I on LBAmp100
      • → 37°C O/N
  3. Re-plate transformants
    • spread remaining 450μL of NovaBlue transformations from yesterday
      • → 37°C O/N
  4. Ligation
    1. 8.5μL ddV(His) from last week + 4μL ddI from last week + 2μL Ligase buffer + 4.5μL sterile H2O + 1μL Muratore:Materials/DNA ligase
    2. 8.5μL ddV from last week + 4μL ddI from last week + 2μL Ligase buffer + 4.5μL sterile H2O + 1μL Muratore:Materials/DNA ligase
      • → 16°C O/N

Results

  • Add results here...
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