Difference between revisions of "A Brief Description of What We Will Do"

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(Created page with 'Step 1 - Taking dry DNA from wells 1. Making sure the plates are properly oriented, punch a hole through the foil cover using a pipette - into the well you want the DNA from. Do...')
 
 
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Step 1 - Taking dry DNA from wells
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'''[[Step 1 - Taking dry DNA from wells]]'''
  
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1. Making sure the plates are properly oriented, punch a hole through the foil cover using a pipette - into the well you want the DNA from. Don't remove foil cover since it might pose the threat of cross-contamination between wells.
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'''[[Step 2 - Transforming competent cells.]]'''
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''Competent cells are those cells which have the ability to take up extracellular/naked DNA from its environment.''
  
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2. Add 15uL of deionized water - diH2O
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'''Step 3 - Pick a single colony.'''
  
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Step 2 - Transforming competent cells. Competent cells are those cells which have the ability to take up extracellular/naked DNA from its environment.
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'''Step 4 - Inoculate broth with Ampicillin-R''' (an antibiotic) and let it grow for 18 hours.
  
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To transform competent cells, we require resuspended DNA and some competent cells.
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'''Step 5 - Use the resulting culture for [[mini-prep.]]''' - ''a process used to purify plasmids and yields clean, usable DNA.''
  
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1. Start thawing the competent cells on crushed ice.
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'''Step 6 - [[Digesting the DNA]]'''
  
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2. Add 50 µL of thawed competent cells and then 1 - 2 µL of the resuspended DNA to the labelled tubes. Make sure to keep the    competent cells on ice.
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'''Step 7 - [[Purifying the DNA]]'''
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3. Incubate the cells on ice for 30 minutes.
 
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4. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.
 
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5. Incubate the cells on ice for 5 minutes.
 
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6. Add 200 μl of LB broth (make sure that the broth does not contain antibiotics and is not contaminated)
 
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7. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency.
 
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8. 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. This helps ensure that you will be able to pick out a single colony.
 
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9. Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics, especially ampicillin, start to break down and un-transformed cells will begin to grow.
 

Latest revision as of 18:59, 16 June 2009

Step 1 - Taking dry DNA from wells

Step 2 - Transforming competent cells. Competent cells are those cells which have the ability to take up extracellular/naked DNA from its environment.

Step 3 - Pick a single colony.

Step 4 - Inoculate broth with Ampicillin-R (an antibiotic) and let it grow for 18 hours.

Step 5 - Use the resulting culture for mini-prep. - a process used to purify plasmids and yields clean, usable DNA.

Step 6 - Digesting the DNA

Step 7 - Purifying the DNA

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