Team:UNC Chapel Hill/4 June 2009

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[[Team:UNC_Chapel_Hill/Notebook|Back]]
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This what we did in Scott's lab on 6/4/09:
This what we did in Scott's lab on 6/4/09:
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===Terminology===
===Terminology===
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Chemically competent E. Coli
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*Chemically competent E. Coli
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Electrocompetent E. Coli
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*Electrocompetent E. Coli
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Electroporation
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*Electroporation
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Plasmid
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*Plasmid
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Ampicillin
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*Ampicillin
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Heat Shock
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*Heat Shock
===Gist of Lab===
===Gist of Lab===
Have six different groups:
Have six different groups:
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{| border="1"
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!  Plasmid
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!  Chemically Competent
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!  Electrocompetent
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|-
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| iGem BBa_I13521
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| Group 1
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| Group 4
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|-
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| pBluescript (Positive Control)
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| Group 2
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| Group 5
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|-
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| No Plasmid
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| Group 3
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| Group 6 
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|}
# - Chemically Competent E. Coli, with iGem BBa_I13521 Plasmid (Experimental group)
# - Chemically Competent E. Coli, with iGem BBa_I13521 Plasmid (Experimental group)
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*Keep Microcentrifuge tubes on ice unless indicated otherwise!
*Keep Microcentrifuge tubes on ice unless indicated otherwise!
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#  Find the iGem part of interest.  Puncture the appropriate well and inject 15 µL of dH20.  Let sit for a couple of minutes to homogenize.  The liquid will turn red due to an indicator in the well.
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#  Found the iGem part of interest (iGem BBa_I13521).  It has a promoter, RBSs, terminators, and a RFP (Red Fluorescent Protein)  Punctured the appropriate well (O6 on the 2nd Set) and injected 15 µL of dH20.  Let sit for a couple of minutes to homogenize.  The liquid turned red due to an indicator in the well.
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# Label 6 Microcentrifuge Tubes numbers 1-6 and sit them in ice.  It's important for them to stayed cold, so that when the bacteria is transferred, the bacteria will not be heat shocked or damaged.
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# Labeled six Microcentrifuge Tubes numbers 1-6 and sat them in ice.  It's important for them to stay cold, so that when the bacteria is transferred, the bacteria will not be damaged.
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#  Find tubes for bacteria of interest from the freezer, in this case: DH5α-E Chemically competent bacteria [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Transformation/Chemically-Competent.html] and Electocompetent bacteria
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#  Found microcentrifuge tubes for bacteria of interest from the freezer, in this case: DH5α-E Chemically competent bacteria [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Transformation/Chemically-Competent.html] and Electocompetent bacteria
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#  Let the bacteria thaw in the ice until a slurry.  Place 25 µL of bacteria into the appropriate tube (CC in 1 through 3; EC in 4 through 6)
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#  Let the bacteria thaw in the ice until a slurry.  Placed 25 µL of bacteria into the appropriate tube (CC in 1 through 3; EC in 4 through 6)
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#  Inject 1 µL of iGem part liquid into the appropriate tubes (1 and 4).  Likewise for the pBluescript (2 and 5)
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#  Added 1 µL of iGem part liquid into the appropriate tubes (1 and 4).  Likewise for the pBluescript (2 and 5)
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#  Let the CC cells sit for 10 minutes.  Afterward, heat shock the tubes at 40 degrees Celsius for 30 seconds each.  Then add 500 µL of broth to each tube and put back onto ice.
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#  Let the CC cells sit for 10 minutes.  Afterward, heat shocked the tubes at 42 degrees Celsius for 30 seconds each.  Then add 500 µL of SOC growth medium to each tube and put back onto ice.
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#  Electroporate 4-6 at 50 µF, 150 ohms, 1.5 kVolts by pulsing twice.  Immediately add 500 µL of broth after shocking and place back onto ice.
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#  Placed 4-6 into individual electroporation cuvettes.  Electroporated tubes 4-6 at 50 µF, 150 ohms, 1.5 kVolts by pulsing twice.  Immediately add 500 µL of SOC growth medium to each after shocking and place back onto ice.
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#  Incubate all the tubes in a shaker for about 1 hour at 37 degrees Celsius.
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#  Incubated all the tubes in a shaker for about 1 hour at 37 degrees Celsius.
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#  Plate 50 µL of bacteria on a corresponding agar dish with ampicillin.
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#  Plated 50 µL of bacteria on a corresponding agar dish with ampicillin or carbenicillin.  Spread around with curved glass pipets.
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#  Incubate overnight in the oven.  Check the colonies the next day.
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#  Incubated overnight in the oven.  Will check the colonies the next day to see if iGem plasmid was taken up.

Latest revision as of 21:27, 12 June 2009

Back

This what we did in Scott's lab on 6/4/09:

Contents

Goals of Lab

  • Learn how to do basic DNA transformation of E. Coli using plasmids
  • Trying using the iGem parts, by putting Part BBa_I13521 into E. Coli [http://partsregistry.org/Part:BBa_I13521]


Terminology

  • Chemically competent E. Coli
  • Electrocompetent E. Coli
  • Electroporation
  • Plasmid
  • Ampicillin
  • Heat Shock

Gist of Lab

Have six different groups:

Plasmid Chemically Competent Electrocompetent
iGem BBa_I13521 Group 1 Group 4
pBluescript (Positive Control) Group 2 Group 5
No Plasmid Group 3 Group 6
  1. - Chemically Competent E. Coli, with iGem BBa_I13521 Plasmid (Experimental group)
  2. - Chemically Competent E. Coli, with pBluescript Plasmid (Positive Control)
  3. - Chemically Competent E. Coli with no plasmid (Negative Control)
  4. - Electrocompetent E. Coli with iGem BBa_I13521 Plasmid (Experimental group)
  5. - Electrocompetent E. Coli with pBluescript Plasmid (Positive Control)
  6. - Electrocompetent E. Coli with no plasmid (Negative Control)

pBluescript is a plasmid that Scott has that is known to work and make the cells white in Bluewhite screening [http://en.wikipedia.org/wiki/PBluescript]. Therefore 2 and 5 are our positive controls. 3 and 6 have no plasmids and are our negative controls. 1 and 4 are our experimental groups because we want to try see what happens by transforming the iGem plasmid using the same procedure.

Procedure

Notes:

  • Keep Microcentrifuge tubes on ice unless indicated otherwise!
  1. Found the iGem part of interest (iGem BBa_I13521). It has a promoter, RBSs, terminators, and a RFP (Red Fluorescent Protein) Punctured the appropriate well (O6 on the 2nd Set) and injected 15 µL of dH20. Let sit for a couple of minutes to homogenize. The liquid turned red due to an indicator in the well.
  2. Labeled six Microcentrifuge Tubes numbers 1-6 and sat them in ice. It's important for them to stay cold, so that when the bacteria is transferred, the bacteria will not be damaged.
  3. Found microcentrifuge tubes for bacteria of interest from the freezer, in this case: DH5α-E Chemically competent bacteria [http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/Transformation/Chemically-Competent.html] and Electocompetent bacteria
  4. Let the bacteria thaw in the ice until a slurry. Placed 25 µL of bacteria into the appropriate tube (CC in 1 through 3; EC in 4 through 6)
  5. Added 1 µL of iGem part liquid into the appropriate tubes (1 and 4). Likewise for the pBluescript (2 and 5)
  6. Let the CC cells sit for 10 minutes. Afterward, heat shocked the tubes at 42 degrees Celsius for 30 seconds each. Then add 500 µL of SOC growth medium to each tube and put back onto ice.
  7. Placed 4-6 into individual electroporation cuvettes. Electroporated tubes 4-6 at 50 µF, 150 ohms, 1.5 kVolts by pulsing twice. Immediately add 500 µL of SOC growth medium to each after shocking and place back onto ice.
  8. Incubated all the tubes in a shaker for about 1 hour at 37 degrees Celsius.
  9. Plated 50 µL of bacteria on a corresponding agar dish with ampicillin or carbenicillin. Spread around with curved glass pipets.
  10. Incubated overnight in the oven. Will check the colonies the next day to see if iGem plasmid was taken up.
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