IGEM:MIT/2007/Notebook/2007-6-14: Difference between revisions

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Contents    Volume    Label
Contents    Volume    Label
U V        (2 µL)    I
U V        (2 µL)    I
dV          (5 µL)    A
ddV        (5 µL)    A
dV + dI    (5 µL)    B
ddV + ddI  (5 µL)    B
ddV        (5 µL)    C
dV          (5 µL)    C
ddV + ddI  (5 µL)    D
dV + dI    (5 µL)    D
dV          (10 µL)  E
ddV        (10 µL)  E
dV + dI    (10 µL)  F
ddV + ddI  (10 µL)  F
ddV        (10 µL)  G
dV          (10 µL)  G
ddV + ddI  (10 µL)  H
dV + dI    (10 µL)  H
</pre><br>
</pre><br>


===Transformation Protocol===
===Transformation Protocol, adapted from OWW===
 
# Thaw TSS cells on ice.
#Aliquot 5 µL and 10 µL of ligation reactions into eppendorfs
# Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
#*For the undigested vector, dilute 100x so we can put in around 2 ng (because optical density of undigested vector is 86.4 ng/µL)
#* If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
#Pipette 50 µL of competent DH5a E. coli bacteria into ligation reaction plasmid eppendorfs from above and mix by tapping gently
#* If you are using the competent cells TK gave us, the volume of DNA added should be < 5% cell volume
#*Do not pipette up and down
# Let sit for 30 minutes on ice.
#Incubate on ice for 30 minutes
#* Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
#Heat shock the bacteria in a 37°C water bath for 20 seconds
# Incubate cells for 45-50 seconds at 42C.
#Add 950 µL LB to each of the eppendorf tubes
# Incubate cells on ice for 2 min.
#Incubate for an hour on a shaker in the 37°C room
# Add 1 mL LB at room temp.
#Centrifuge at 8000 rpm for 5 minutes
# Incubate for 1 hour at 37oC on shaker.
#Decant 900 uL of supernatant and resuspend the rest of the cells
#*Note: tetR and cmR need 1.5 - 2 hrs
#Take the contents of tubes and spread them on LB Amp plates
# Spread 100-300 μl onto a plate made with appropriate antibiotic.
#*B has a hole, G is barbequed toast, literally
# Grow overnight at 37 °C.
# Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.




Experiment notes
*B has a hole, G is barbequed toast, literally
*1:10am (13 hrs incubation): only plates C,D,H,I had small colonies.
*1:10am (13 hrs incubation): only plates C,D,H,I had small colonies.
**plate I had most colonies (~15)
**plate I had most colonies (~15)

Latest revision as of 09:18, 18 July 2007

Grad Advisors

  • Morning: Debbie, Forrest
  • Afternoon: Brian, Forrest, Robbie, L-A
  • Late Night 1:10am: Debbie, Forrest

LAB WORK

  • Make competent E. coli cells
  • Transformation into competent cells
  • Plate transformed cells


Depending on how fast cells grow......

  • Night time
    • Analyze plates
    • innoculate 4ml LB/Amp

Procedures

Details of DH5 Strain E Coli

Regular cell membranes are not permeable to plasmids, so we much use ions (CaCl) to make the bacteria competent and able to accept foreign DNA.
Start: 2 vials of competent bacteria (500 µL each)
9 Transformations

Contents    Volume    Label
U V         (2 µL)    I
ddV         (5 µL)    A
ddV + ddI   (5 µL)    B
dV          (5 µL)    C
dV + dI     (5 µL)    D
ddV         (10 µL)   E
ddV + ddI   (10 µL)   F
dV          (10 µL)   G
dV + dI     (10 µL)   H


Transformation Protocol, adapted from OWW

  1. Thaw TSS cells on ice.
  2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
    • If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
    • If you are using the competent cells TK gave us, the volume of DNA added should be < 5% cell volume
  3. Let sit for 30 minutes on ice.
    • Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
  4. Incubate cells for 45-50 seconds at 42C.
  5. Incubate cells on ice for 2 min.
  6. Add 1 mL LB at room temp.
  7. Incubate for 1 hour at 37oC on shaker.
    • Note: tetR and cmR need 1.5 - 2 hrs
  8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
  9. Grow overnight at 37 °C.
  10. Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.


Experiment notes

  • B has a hole, G is barbequed toast, literally
  • 1:10am (13 hrs incubation): only plates C,D,H,I had small colonies.
    • plate I had most colonies (~15)
    • all others had around 1-8 colonies

Project Research Homework

  • Aditya: sensory promotors / metal ion binding
  • Alex, Bernice: past iGEM projects (2006, 2005)
  • Semmie,Jessica: Hwang papers (mussel proteins)
  • Toan: Rice07 (read for bacterial display)
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