Team:OU-Norman OK/Project/Notebook
From 2013.igem.org
03/13/13
Making and Preparing Agarose Gel
- In a 125mL erlenmyer flask, combine 0.4g ultra pure agarose and 40mL 1x TAE buffer, which makes a 1% gel
- Swirl to mix. Place in microwave for 40 seconds. If all agarose isn't dissolved, heat again in 7 second increments
- Run flask bottom under water to cool agarose
- Pour into gel rig with comb inserted
- Let gel cool until opaque
Running Gel of Post-Digested pSB1C3 and pSB1A3
- Move gel into gel rig container, pour 1x TAE buffer until it covers the gel surface
- Remove comb slowly
- Mix 5-10μL of digest with 3μL of 1:4 EZ Vision dye (Note: if using undigested DNA, only use 2μL
- Load samples into wells after 1 kb DNA ladder is loaded into far left lane
- well 1:Ladder
- well 2:pSB1C3 EcoR1
- well 3:pSB1C3 EcoR1 Pst1
- well 4:pSB1C3 Pst1
- well 5:pSB1A3 EcoR1
- well 6:pSB1A3 EcoR1 Pst1
- well 7:pSB1A3 Pst1
- well 8:pSB1C3 UNDIGESTED
- Run gel for 45 minutes-1.5 hours at 85 volts
03/12/13
Preformed the following restriction digest of pSB1A3 and pSB1C3.
500ng DNA in 20μL
pSB1A3 [DNA] = 135.8ng/μL
pSB1C3 [DNA] = 74.7ng/μL
500ng x μL/135.8μg = 3.68μL
500ng x μL/74.7μg = 6.69μL
Sample | EcoRI | PstI | 10X Buffer | DNA | PCR Water | TOTAL |
---|---|---|---|---|---|---|
pSB1A3 | 2μL | 0μL | 2μL | 4μL | 12μL | 20μL |
pSB1A3 | 2μL | 2μL | 2μL | 4μL | 10μL | 20μL |
pSB1C3 | 2μL | 0μL | 2μL | 7μL | 9μL | 20μL |
pSB1C3 | 2μL | 2μL | 2μL | 7μL | 7μL | 20μL |
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03/8/13
Protocol
- Open Nanodrop 7000 V.6.0
- Select Nucleic Acid
- Blank via 3μL of PCR water
- Verify 0ng/&#;L DNA in PCR water
- Load 3μL of sample
- Record DNA concentration in ng/μL
- Print Screen
pSB1K3
p34KM
pIKM1
pSB1A3
pSB1C3
Back to top02/13/13
Colonies were counted
1:1000 dilution of AmpicillinR pSB1A3 wasn't properly plated
Dilution | Plasmid | Colony Count | Cells/μg DNA |
---|---|---|---|
1:1000 | pSB1C3 | 58 | 8.24e6 |
1:1000 | pSB1K3 | 79 | 1.12e7 |
1:1000 | p34KM | 157 | 2.22e7 |
1:100 | pSB1A3 | 1521 | 2.16e7 |
Note: 1:100 estimates by counting colonies in 1/3 of plate and multiplying by 3
Back to top02/12/13
Plates were removed from 37°C incubator, wrapped in parafilm, and stored in 4°C refrigerator overnight
Back to top02/13/13
pSB1C3
Dilution 1:1000
58 Colonies
Back to top02/11/13
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
- TOP10 competent cells in 100μL alliquots (x5) were thawed on ice and resuspended.
- 100ng of plasmid were added to cells
- Cells were placed on ice for 20 minutes
- Cells were transformed to a 42°C waterbath for 60 seconds
- After 60 seconds in waterbath, add 600μL of Psi proth IMMEDIATELY to clls
- Cells were incubated for 60 minutes at 37°C while shaking at 200rpm
- Dilutions of transformation mixture were made at 1:10, 1:100, and 1:1000
- 50mL of each dilution was plated on an LB + Antibiotic plate
- Plates were incubated at 37°C overnight
Plasmids were diluted to 20μL of 10μg/μL
p34KM
pSB1C3
pSB1A3
pSB1K3
Back to topBuffers 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 |
|
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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
Back to top01/25/13
Restreaked TOP10 cells on LB plates for isolation of single colonies.
Back to top01/23/13
Sealed TOP10 E. coli cells on LB agar plate. Stored in 37°C incubator
Back to top01/18/13
Poured LB agar plates
Back to top01/16/13
Making Agar Plates
LB agar is used to grow our stocks of Escherichia coli
- 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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