Kit to Stock Plasmid

From 2010.igem.org

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KIT TO STOCK
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'''EXTRACTION FROM KIT'''
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1. With a pipette tip, punch a hole through the foil cover into the corresponding well to the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells.  
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#With a pipette tip, punch a hole through the foil cover into the corresponding well to the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells.  
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2. Add 10uL of diH2O (deionized water)  
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#Add 10uL of diH2O (deionized water)  
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3. Pipette 1 or 2uL of the resuspended DNA Transformation into your desired competent cells, plate bacteria with the appropriate antibiotic* and grow overnight.  
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#Pipette 1 or 2uL of the resuspended DNA Transformation into your desired competent cells, plate bacteria with the appropriate antibiotic* and grow overnight.  
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Transformation
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'''TRANSFORMATION'''
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1. Thaw cells on ice and pipette 25uL aliquots into tubes on ice
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2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
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o Note: If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
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3. Let sit for 5 minutes on ice.
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o Note: For better efficiency do at least 30 minutes
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4. Incubate cells for 45 seconds at 42C.
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5. Incubate cells on ice for 2 min.
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6. Add 200 uL LB (2XYT and SOC are also suitable for greater efficiency)
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7. Incubate for 1 hour at 37C (for amp you can incubate for 5-10 minutes if you are in a hurry, for other ABs, can probably get away with ~40 minutes)
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8. Spread 100-300 μl onto a plate made with appropriate antibiotic.
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9. Grow overnight at 37C.
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10. Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.
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#Thaw cells on ice and pipette 25uL aliquots into tubes on ice
 +
#Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
 +
#* If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
 +
#Let sit for 5 minutes on ice.
 +
#* For better efficiency do at least 30 minutes
 +
#Incubate cells for 45 seconds at 42C.
 +
#Incubate cells on ice for 2 min.
 +
#Add 200 uL LB (2XYT and SOC are also suitable for greater efficiency) --> no antibiotic
 +
#Incubate for 1 hour at 37C (for amp you can incubate for 5-10 minutes if you are in a hurry, for other ABs, can probably get away with ~40 minutes)
 +
#Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance.
 +
#Plate 20 µl and 200 µl of the transformation onto the dishes and spread.
 +
#Incubate at 37C for 16 hours.
 +
#Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.
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4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 18 hours.  
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#Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 18 hours.  
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5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.  
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#Use the resulting culture to miniprep the DNA AND make your own glycerol stock
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#* We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.  
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#* See Miniprep Manual
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See Miniprep Manual
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'''GLYCEROL STOCK'''
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#Add 1 ml of 40% glycerol in H2O to a cryogenic vial.
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#Add 1 ml sample from the culture of bacteria to be stored.
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#Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
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#*Alternatively, pipet to mix.
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#Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
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#On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
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#Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later.
Back to [[Team:Northwestern/Protocol|<span style="color:#4B088A;">Protocol</span>]]
Back to [[Team:Northwestern/Protocol|<span style="color:#4B088A;">Protocol</span>]]
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[[Category:Protocol]]

Latest revision as of 19:19, 16 October 2010

EXTRACTION FROM KIT

  1. With a pipette tip, punch a hole through the foil cover into the corresponding well to the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells.
  2. Add 10uL of diH2O (deionized water)
  3. Pipette 1 or 2uL of the resuspended DNA Transformation into your desired competent cells, plate bacteria with the appropriate antibiotic* and grow overnight.

TRANSFORMATION

  1. Thaw cells on ice and pipette 25uL aliquots into tubes on ice
  2. Add DNA, pipette gently to mix (1μl of prepped plasmid is more than enough).
    • If you are adding small volumes (~1μl), be careful to mix the culture well. Diluting the plasmid back into a larger volume can also help.
  3. Let sit for 5 minutes on ice.
    • For better efficiency do at least 30 minutes
  4. Incubate cells for 45 seconds at 42C.
  5. Incubate cells on ice for 2 min.
  6. Add 200 uL LB (2XYT and SOC are also suitable for greater efficiency) --> no antibiotic
  7. Incubate for 1 hour at 37C (for amp you can incubate for 5-10 minutes if you are in a hurry, for other ABs, can probably get away with ~40 minutes)
  8. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance.
  9. Plate 20 µl and 200 µl of the transformation onto the dishes and spread.
  10. Incubate at 37C for 16 hours.
  11. Save the rest of the transformants in liquid culture at 4 °C. If nothing appears on your plate, you can spin this down, resuspend in enough medium to spread on one plate and plate it all. This way you will find even small numbers of transformants.
  1. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 18 hours.
  2. Use the resulting culture to miniprep the DNA AND make your own glycerol stock
    • We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.
    • See Miniprep Manual

GLYCEROL STOCK

  1. Add 1 ml of 40% glycerol in H2O to a cryogenic vial.
  2. Add 1 ml sample from the culture of bacteria to be stored.
  3. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed.
    • Alternatively, pipet to mix.
  4. Use a tough spot to put the name of the strain or some useful identifier on the top of the vial.
  5. On the side of the vial list all relevant information - part, vector, strain, date, researcher, etc.
  6. Store in a freezer box in a -80C freezer. Remember to record where the vial is stored for fast retrieval later.

Back to Protocol

Retrieved from "http://2010.igem.org/Kit_to_Stock_Plasmid"