Team:OU-Norman OK/Project/Notebook

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<p class = "date">02/12/13</p>
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<p>Plates were removed from 37&deg;C incubator, wrapped in parafilm, and stored in 4&deg;C refrigerator overnight</p>
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<p>50mL of stock were prepared and split into several 15mL tubes</p>
<p>50mL of stock were prepared and split into several 15mL tubes</p>
<p>Stocks should be stored in the refrigerator at 4&#176;C</p>
<p>Stocks should be stored in the refrigerator at 4&#176;C</p>
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<p class="date">01/25/13</p>
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<p>Restreaked TOP10 cells on LB plates for isolation of single colonies.</p>
<p>Restreaked TOP10 cells on LB plates for isolation of single colonies.</p>
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<p class = "date">01/23/13</p>
<p class = "date">01/23/13</p>
<p>Sealed TOP10 E. coli cells on LB agar plate. Stored in 37&#176;C incubator</p>
<p>Sealed TOP10 E. coli cells on LB agar plate. Stored in 37&#176;C incubator</p>
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<p class = "date">01/18/13</p>
<p class = "date">01/18/13</p>
<p>Poured LB agar plates</p>
<p>Poured LB agar plates</p>
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<p>Mix Components in 1L of dH<sub>2</sub>O until dissolved. Spilt 1000mL solution into two flask. Cap flask with aluminum to prevent spilling of solution. Autoclave on slow exhaust for 20 minutes. Keep liquid in 65&#176;C water bath to prevent setting until 18-Jan. </p>
<p>Mix Components in 1L of dH<sub>2</sub>O until dissolved. Spilt 1000mL solution into two flask. Cap flask with aluminum to prevent spilling of solution. Autoclave on slow exhaust for 20 minutes. Keep liquid in 65&#176;C water bath to prevent setting until 18-Jan. </p>
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Revision as of 16:55, 8 August 2013

02/12/13

Plates were removed from 37°C incubator, wrapped in parafilm, and stored in 4°C refrigerator overnight


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02/13/13

pSB1C3

Dilution 1:1000

58 Colonies


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02/11/13

Transforming Competent Cells

We transformed TOP10 chemically competent cells using plasmids.


Resistance Plasmid ID Original Concentration (ng/μL)
Kanamycin pSB1K3 62
Kanamycin p34KM 40
Chloramphenicol pSB1C3 43
Ampicillin pSB1A3 75

Protocol

  1. TOP10 competent cells in 100μL alliquots (x5) were thawed on ice and resuspended.
  2. 100ng of plasmid were added to cells
  3. Cells were placed on ice for 20 minutes
  4. Cells were transformed to a 42°C waterbath for 60 seconds
  5. After 60 seconds in waterbath, add 600μL of Psi proth IMMEDIATELY to clls
  6. Cells were incubated for 60 minutes at 37°C while shaking at 200rpm
  7. Dilutions of transformation mixture were made at 1:10, 1:100, and 1:1000
  8. 50mL of each dilution was plated on an LB + Antibiotic plate
  9. Plates were incubated at 37°C overnight


Plasmids were diluted to 20μL of 10μg/μL

p34KM

10ng/μL×μL/62ng×20μL = 3.2μL (original plasmid concentration) + 16.8μL (dH2O)

pSB1C3

10ng/μL×μL/43ng×20μL = 4.65μL (original plasmid concentration) + 15.35μL (dH2O)

pSB1A3

10ng/μL×μL/75ng×20μL = 2.6μL (original plasmid concentration) + 17.4μL (dH2O)

pSB1K3

10ng/μL×μL/40ng×20μL = 5.0μL (original plasmid concentration) + 15.0μL (dH2O)

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Buffers for Preparing Competent E. coli

02/4/13

TFB1: pH: 5.8/ Sterile Filter


Chemicals Concentration (mM)
RbCl 100
MnCl2 50
Potassium Acetate 30
CaCl2 10
Glycerol 15% by Weight

TFB2 ph:6.8 (Use KOH to adjust)/ Sterile Filter

Chemicals Concentrations (mM)
MOPS 10
RbCl 10
CaCl2 75
Glycerol 15% by Weight

TFB1
Chemicals Mass (g)
RbCl 3.02965
MnCl2 1.56996
Potassium Acetate 0.74392
CaCl2 0.27806
Glycerol 37.5463

TFB2
Chemicals Mass (g)
MOPS 0.53398
RbCl 0.30225
CaCl2 2.88320
Glycerol 37.5457
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Making Antibiotic Stocks

02/1/13

Antibiotics are used to isolate organisms into which plasmids containing antibiotic resistance genes have been transformed.

Make antibiotic plates with the following specs.


Antibiotic Concentration (µg/mL) Color Code
Ampicillin 100 Orange
Chloramphenicol 35 Green
Kanamycin 50 Red
Tetracycline 15 Yellow

Stocks should be made at 1000x concentration, so that making 1L of plate medium will require only 1mL of stock antibiotic solution.


Stocks of antibiotics are made at the following concentrations


Ampicillin 100 mg/mL
Chloramphenicol 35 mg/mL
Kanamycin 30 mg/mL
Tetracycline 15 mg/mL

50mL of stock were prepared and split into several 15mL tubes

Stocks should be stored in the refrigerator at 4°C


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01/25/13

Restreaked TOP10 cells on LB plates for isolation of single colonies.


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01/23/13

Sealed TOP10 E. coli cells on LB agar plate. Stored in 37°C incubator


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01/18/13

Poured LB agar plates


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01/16/13

Making Agar Plates

LB agar is used to grow our stocks of Escherichia coli


Recipe: Per 1000 mL
  • 10g Bacto Tryptone
  • 5g Yeast Extract
  • 10g Sodium Chloride (NaCl)
  • 15g Agarose

Mix Components in 1L of dH2O until dissolved. Spilt 1000mL solution into two flask. Cap flask with aluminum to prevent spilling of solution. Autoclave on slow exhaust for 20 minutes. Keep liquid in 65°C water bath to prevent setting until 18-Jan.


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