Team:Virginia/Protocols

From 2008.igem.org

(Difference between revisions)
(→Gel Electrophoresis (Making the gel))
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==DNA Elution==
==DNA Elution==
*Obtain Parts binder and confirm desired Plate and Well number
*Obtain Parts binder and confirm desired Plate and Well number
-
*Warm 10µL aliquot of 10:1 TE Buffer pH 8.0 in 1.5mL labeled microcentrifuge tube to 50°C for each desired part
+
*Warm a 10µL aliquot of 10:1 TE Buffer pH 8.0 in 1.5mL labeled microcentrifuge tube in the dry heating block to 50°C for each desired part
*Clean punch tool
*Clean punch tool
**Punch clean thesis paper (remember cutting pad) and discard resulting chad
**Punch clean thesis paper (remember cutting pad) and discard resulting chad
Line 84: Line 84:
**Once filter paper is cut, use pusher end of punch tool to eject chad into the corresponding labeled tube of TE Buffer
**Once filter paper is cut, use pusher end of punch tool to eject chad into the corresponding labeled tube of TE Buffer
*Clean punch tool and repeat for each desired part
*Clean punch tool and repeat for each desired part
-
*Let sit for 20 minutes at 50°C
+
*Let sit for 20 minutes at 50°C in the dry heating block
*Centrifuge at 13,000 rpm for 3 minutes to maximize DNA concentration in solution
*Centrifuge at 13,000 rpm for 3 minutes to maximize DNA concentration in solution
 +
*Keep unused eluted DNA in 4°C fridge
==Cell Transformation==
==Cell Transformation==
-
 
+
*If retrieving previously eluted DNA from the fridge, let sit 20 minutes at 50°C in dry heating block
 +
*Obtain bucket of ice
 +
*Retrieve competent cells from -80°C freezer and place immediately on ice
 +
*Transform the cells
 +
**Label a 1.5mL microcentrifuge tube for each desired transformation and place on ice
 +
**Pipette 25μL of thawed cells into each tube
 +
**Pipette 5μL eluted DNA into each tube, inserting under the surface of the cells
 +
**Let sit on ice for 30 minutes (put unused cells and DNA back)
 +
**Heat shock in 42°C water bath for 1 minute if DH5α, 45 seconds if DB3.1
 +
**Let sit on ice for 2 minutes
 +
**Add 225μL of SOC Medium (check for turbidity before use) to each tube
 +
**Place in agitating incubator and let shake at 37°C for 2 hours
 +
*Remove plates with corresponding antibiotics from fridge well in advance of plating so they're room temperature
 +
*Plate each tube of cells
 +
**Dip glass spreader in 95% ethanol and burn on Bunsen burner, giving plenty of time to cool completely
 +
**Pipette 200μL from each tube onto labeled plate
 +
**Spread over surface of plate with sterile glass spreader
 +
**Resterilize spreader and repeat with the rest of the tubes
 +
**Place the plates LB down in 37° incubator (not shaking) for 5 minutes
 +
**Turn the plates over (to ensure condensation doesn't kill the cells) and incubate overnight
==DNA Digestion==
==DNA Digestion==

Revision as of 14:35, 30 July 2008

Contents

QIAGEN Mini-Prep

  • Spin cell broth down in centrifuge at >13200 revolutions per minute in microcentrifuge (generally 4 minutes is adequate).
  • Pour off remaining broth and resuspend in 250 uL of Buffer P1 (LyseBlue is nice to have).
  • Add 250 uL Buffer P2 (for cell lysis). Invert microcentrifuge tubes 4-6 times. Do NOT allow to run for more than 5 minutes.
  • Add 350 uL Buffer N3 and invert tubes another 4-6 times.
  • Spin in microcentrifuge at >13000 revolutions per minute for ten minutes.
  • Transfer supernatant to provided spin column.
  • Run spin column in microcentrifuge for 60 seconds. Discard flow-through.
  • Add 0.5 mL Buffer PB. Centrifuge for 60 seconds. Discard flow-through.
  • Add 0.75 mL Buffer PE. Centrifuge for 60 seconds. Discard flow through.
  • Centrifuge for an additional 60 seconds.
  • Transfer spin-column top to a microcentrifuge tube for collection.
  • Add 50 uL Buffer EB to center of column. Allow to sit for one minute.
  • Centrifuge for one minute. The clear liquid in the bottom is your DNA solution.


Gel Electrophoresis (Making the gel)

  • In a 100 mL Erlenmeyer flask, place and pour: 0.5 g Agar and 50 mL 1X TAE Buffer
  • Microwave for 30 seconds and then swirl the flask
  • Microwave for 15 seconds (caution: do not let the solution boil out of the flask)
  • Pipette 2 microliters of Ethidium Bromide into the gel solution and then swirl flask
    • Ethidium Bromide stains the plate for DNA to be observed under UV light
  • NOTE: Ethidium Bromide is a hazardous chemical and must be disposed of properly - this includes the gel itself
  • Setup gel holder with desired size and number comb perpendicular leads so that liquid gel stays contained
  • Pour all of hot gel into gel holder, cover with saran wrap and leave out to solidify


  • This is a 1% agar gel; depending on the size of your DNA fragment, you will want to increase concentration of the agar for smaller pieces of DNA
  • for DNA fragments between 10 to 100 basepairs - it is advisable you use specially prepared agar such as nusieve GTG agar which can be obtained online


Gel Electrophoresis (Loading & Running)

  • Align wells on the side of the negetive(black) lead
  • Fill with 1X TAE buffer to cover gel with thin layer of buffer
  • carefully remove the comb
  • Using small epindorf tubes mix together DNA and 6X loading dye with a 6:1 ratio (this ratio is dependant on the loading dye)
  • Mix 1 uL of 1kb ladder with 5 uL of water and 1 uL of 6X loading maintaining the ratio
  • Carefully pipette the DNA mixtures into the wells being careful not to pierce the bottom of the wells


3A assembly Ligation

  • Assuming the 3 parts that are going to be ligated are cut appropriately
    • First part cut at EcoRI and SpeI
    • Second part cut at XbaI and PstI
    • Back bone/Vector cut at EcoRI and PstI
    • this works because S and X form a scar and the other restriction sites are preserved and the ligated insert keeps the same format with EX as a prefix and SP as a suffix
    • the back bones should have a different antibiotic resistance than the 2 inserts to guarantee that any colonies that grow are products of a successful ligation
    • Note: should not be done in DB3.1 cells


  • For 20ul mix:
    • 4 ul water
    • 2 ul T4 10X ligase buffer
    • 6 ul insert 1
    • 6 ul insert 2
    • 1 ul vector
    • 1 ul T4 DNA ligase (enzyme)


  • For 50ul mix:
    • 17 ul water
    • 5 ul T4 10X ligase buffer
    • 12 ul insert 1
    • 12 ul insert 2
    • 2 ul vector
    • 2 ul T4 DNA ligase (enzyme)


DNA Elution

  • Obtain Parts binder and confirm desired Plate and Well number
  • Warm a 10µL aliquot of 10:1 TE Buffer pH 8.0 in 1.5mL labeled microcentrifuge tube in the dry heating block to 50°C for each desired part
  • Clean punch tool
    • Punch clean thesis paper (remember cutting pad) and discard resulting chad
    • Disassemble punch tool into pushrod, casing, and endcap (spring, endcap chamber, endcap lid)
    • Dip pushrod and blade into each stage of cleaning station in sequence: bleach, distilled water, distilled water, 190 proof ethanol
    • Blot clean with Kimwipe
    • Let air dry on Kimwipe for 5 minutes
    • Reassemble punch tool
  • Confirm Plate and Well number
  • Punch part
    • Ensure punch tool is completely dry by tapping out on a Kimwipe!
    • Slide cutting pad (in back of binder) underneath the binder page with your part
    • Push (hard!) the punch tool blade into the corner of the desired filter paper stain, rotating to cut
    • Once filter paper is cut, use pusher end of punch tool to eject chad into the corresponding labeled tube of TE Buffer
  • Clean punch tool and repeat for each desired part
  • Let sit for 20 minutes at 50°C in the dry heating block
  • Centrifuge at 13,000 rpm for 3 minutes to maximize DNA concentration in solution
  • Keep unused eluted DNA in 4°C fridge


Cell Transformation

  • If retrieving previously eluted DNA from the fridge, let sit 20 minutes at 50°C in dry heating block
  • Obtain bucket of ice
  • Retrieve competent cells from -80°C freezer and place immediately on ice
  • Transform the cells
    • Label a 1.5mL microcentrifuge tube for each desired transformation and place on ice
    • Pipette 25μL of thawed cells into each tube
    • Pipette 5μL eluted DNA into each tube, inserting under the surface of the cells
    • Let sit on ice for 30 minutes (put unused cells and DNA back)
    • Heat shock in 42°C water bath for 1 minute if DH5α, 45 seconds if DB3.1
    • Let sit on ice for 2 minutes
    • Add 225μL of SOC Medium (check for turbidity before use) to each tube
    • Place in agitating incubator and let shake at 37°C for 2 hours
  • Remove plates with corresponding antibiotics from fridge well in advance of plating so they're room temperature
  • Plate each tube of cells
    • Dip glass spreader in 95% ethanol and burn on Bunsen burner, giving plenty of time to cool completely
    • Pipette 200μL from each tube onto labeled plate
    • Spread over surface of plate with sterile glass spreader
    • Resterilize spreader and repeat with the rest of the tubes
    • Place the plates LB down in 37° incubator (not shaking) for 5 minutes
    • Turn the plates over (to ensure condensation doesn't kill the cells) and incubate overnight

DNA Digestion

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