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Team:University of Chicago/Notebook/Transformations
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(New page: ==Chemocompetent cells== ===General transformation== #Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) * This is at 10 pg/_l or 10-5 _g/_l * This can be made by ...) |
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==Chemocompetent cells== | ==Chemocompetent cells== | ||
| - | ===General transformation== | + | ===General transformation=== |
#Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) | #Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen) | ||
| - | * This is at 10 pg/ | + | #* This is at 10 pg/microliter or 10-5 _g/_l |
| - | + | #* This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE | |
# Hold on ice 0.5 hours | # Hold on ice 0.5 hours | ||
# Heat shock 60 sec at 42C | # Heat shock 60 sec at 42C | ||
# Add 250 _l SOC | # Add 250 _l SOC | ||
# Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated | # Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated | ||
| - | * using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. | + | #* using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration. |
| - | * For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol | + | #* For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies |
| - | and tetracycline resistant, we find growing for 2 hours yields many more colonies | + | #* Ampicillin and kanamycin appear to do fine with 1 hour growth |
| - | * Ampicillin and kanamycin appear to do fine with 1 hour growth | + | |
# Plate 20 _l on AMP plates using sterile 3.5 mm glass beads | # Plate 20 _l on AMP plates using sterile 3.5 mm glass beads | ||
* Good cells should yield around 100 - 400 colonies | * Good cells should yield around 100 - 400 colonies | ||
* Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA | * Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA | ||
* We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA | * We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA | ||
| + | |||
| + | ===iGEM transformation (using paper spots)=== | ||
| + | '''Materials needed''' | ||
| + | *Spots soaked in 5microliters warm TE for 20 minutes | ||
| + | *2.0 conical bottom tubes | ||
| + | *Ice | ||
| + | *Competent cells | ||
| + | *42C water bath/37C incubator | ||
| + | *SOC--freshly made! | ||
| + | *Petri plates with appropriate antibiotic | ||
| + | *Transformaiton control DNA, like pGreen | ||
| + | '''Procedure''' | ||
| + | #Soak the spots in 5microliters of the warmed TE for 20 minutes. This allows the maximum concentration of DNA in solution. Start thawing the competent cells on wet crushed ice | ||
| + | #Chill lageled 2mL conical bottom tubes on wet ice. Add 2 microliters of DNA in TE into 50microliters thawed TOP10 cells in 2mL tubes. 2mL tubes allow better liquid movement durin incubation. | ||
| + | #*Make sure to do a control transformation! iGEM uses 1microliter of pUC19 plasmid DNA from invitrogen; we'll use pGreen from Dr. Schonbaum | ||
| + | #Hold the DNA and competent cells on ice for 30 mintues. | ||
| + | #Heat shock cells by immersion in a pre-heated water bath at 42C for 60 seconds. A water bath is important to improve heat transfer to the cells. | ||
| + | # Incubate cell son ice for 2 minutes | ||
| + | #Add 200microliters of SOC broth. This should have NO antibiotics. | ||
| + | #Incubate cells at 37C for 2 hours while tubes are rotating/shaking. 2 hours growth helps in transfomation efficiency, especially for plasmids with resistance other than ampicillin. | ||
| + | # LAbel an LB agar plate w/appropriate antibiotic(s) with the part number, plasmid, and resistance. Plate 250 microliters of the cell culture on the plate. | ||
| + | #Incubate plate at 37C ofr 12-14 hours, making sure agar side is up. If incubated for too long, antibiotics, '''especially ampicillin''' to break down and untransformed cells will grow. | ||
Latest revision as of 15:15, 8 August 2008
Chemocompetent cells
General transformation
- Transform 50 _l of cells with 1 _l of standard pUC19 plasmid (Invitrogen)
- This is at 10 pg/microliter or 10-5 _g/_l
- This can be made by diluting 1 _l of NEB pUC19 plasmid (1 _g/_l, NEB part number N3401S) into 100 ml of TE
- Hold on ice 0.5 hours
- Heat shock 60 sec at 42C
- Add 250 _l SOC
- Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
- using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
- For our plasmids (pSB1AC3, pSPAT3) which are chloramphicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
- Ampicillin and kanamycin appear to do fine with 1 hour growth
- Plate 20 _l on AMP plates using sterile 3.5 mm glass beads
- Good cells should yield around 100 - 400 colonies
- Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
- We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA
iGEM transformation (using paper spots)
Materials needed
- Spots soaked in 5microliters warm TE for 20 minutes
- 2.0 conical bottom tubes
- Ice
- Competent cells
- 42C water bath/37C incubator
- SOC--freshly made!
- Petri plates with appropriate antibiotic
- Transformaiton control DNA, like pGreen
Procedure
- Soak the spots in 5microliters of the warmed TE for 20 minutes. This allows the maximum concentration of DNA in solution. Start thawing the competent cells on wet crushed ice
- Chill lageled 2mL conical bottom tubes on wet ice. Add 2 microliters of DNA in TE into 50microliters thawed TOP10 cells in 2mL tubes. 2mL tubes allow better liquid movement durin incubation.
- Make sure to do a control transformation! iGEM uses 1microliter of pUC19 plasmid DNA from invitrogen; we'll use pGreen from Dr. Schonbaum
- Hold the DNA and competent cells on ice for 30 mintues.
- Heat shock cells by immersion in a pre-heated water bath at 42C for 60 seconds. A water bath is important to improve heat transfer to the cells.
- Incubate cell son ice for 2 minutes
- Add 200microliters of SOC broth. This should have NO antibiotics.
- Incubate cells at 37C for 2 hours while tubes are rotating/shaking. 2 hours growth helps in transfomation efficiency, especially for plasmids with resistance other than ampicillin.
- LAbel an LB agar plate w/appropriate antibiotic(s) with the part number, plasmid, and resistance. Plate 250 microliters of the cell culture on the plate.
- Incubate plate at 37C ofr 12-14 hours, making sure agar side is up. If incubated for too long, antibiotics, especially ampicillin to break down and untransformed cells will grow.
