Team:University of Chicago/Notebook/Transformations

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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/microliter or 10-5 _g/_l
#* This is at 10 pg/microliter or 10-5 _g/_l
Line 16: Line 16:
* 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

  1. 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
  2. Hold on ice 0.5 hours
  3. Heat shock 60 sec at 42C
  4. Add 250 _l SOC
  5. 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
  6. 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

  1. 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
  2. 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
  3. Hold the DNA and competent cells on ice for 30 mintues.
  4. 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.
  5. Incubate cell son ice for 2 minutes
  6. Add 200microliters of SOC broth. This should have NO antibiotics.
  7. 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.
  8. 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.
  9. 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.
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