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Team:University of Chicago/Notebook/Transformations
From 2008.igem.org
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*Petri plates with appropriate antibiotic | *Petri plates with appropriate antibiotic | ||
*Transformaiton control DNA, like pGreen | *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. | #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 | #*Make sure to do a control transformation! iGEM uses 1microliter of pUC19 plasmid DNA from invitrogen; we'll use pGreen from Dr. Schonbaum | ||
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 transformatin (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.
