IGEM:Harvard/2007/Protocols

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(<p style="font-size: 150%;">iGEM Protocol for 2007</p>)
(<p style="font-size: 150%;">iGEM Protocol for 2007</p>)
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*Day 3: DNA Plasmid Preps
*Day 3: DNA Plasmid Preps
#Take out the 6 tubes of grown bacteria in liquid medium and spin two tubes with 1 mL in each for 1 minute at high speed.
#Take out the 6 tubes of grown bacteria in liquid medium and spin two tubes with 1 mL in each for 1 minute at high speed.
-
#Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge
+
#Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
-
tube.
+
##Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
-
##Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible
+
##If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
-
after resuspension of the pellet.
+
-
##If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle
+
-
to ensure LyseBlue particles are completely dissolved. The bacteria should be
+
-
resuspended completely by vortexing or pipetting up and down until no cell clumps
+
-
remain.
+
#Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
#Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
-
##Mix gently by inverting the tube. Do not vortex, as this will result in shearing of
+
##Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
-
genomic DNA. If necessary, continue inverting the tube until the solution becomes
+
##If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.
-
viscous and slightly clear. Do not allow the lysis reaction to proceed for more than
+
#Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
-
5 min.
+
##To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to 10 times. The solution should become cloudy.
-
##If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition
+
##If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.
-
of Buffer P2. Mixing should result in a homogeneously colored suspension. If
+
#Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
-
the suspension contains localized colorless regions or if brownish cell clumps are
+
##A compact white pellet will form.
-
still visible, continue mixing the solution until a homogeneously colored suspension
+
#Apply the supernatants that resulted from step 4 to the QIAprep spin column by decanting or pipetting.
-
is achieved.
+
#Centrifuge for a minute. Discard the flow-through.
-
#Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube
+
#Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for a minute. Discard the flow-through.
-
4–6 times.
+
##This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α™ do not require this additional wash step.
-
##To avoid localized precipitation, mix the solution thoroughly, immediately after
+
#Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for a minute.  Discard flow-through.
-
addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up
+
#Centrifuge for an additional 1 min to remove residual wash buffer.
-
to 10 times. The solution should become cloudy.
+
##Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
-
##If LyseBlue reagent has been used, the suspension should be mixed until all trace
+
#Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
-
of blue has gone and the suspension is colorless. A homogeneous colorless suspension
+
-
indicates that the SDS has been effectively precipitated.
+
-
 
+
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<p style="text-align: right;">Protocol Recorded by Kevin Shee</p>
<p style="text-align: right;">Protocol Recorded by Kevin Shee</p>

Revision as of 14:43, 13 June 2007

iGEM Protocol for 2007

  • Day 1: Transformation protocol (6/11/07)
  1. Thaw the 6 tubes of cells on ice and mix gently to ensure that the cells are evenly suspended.
    1. OMPA1 in PET
    2. OMPA1 + his in PET
    3. OMPA1 + strep2 in PET
    4. OMPA2 in PET
    5. OMPA2 + his in PET
    6. OMPA2 + strep in PET
  2. Add 1 µl of the DNA solution directly to the cells. Stir gently to mix.
  3. Place the tubes on ice for a little more than 5 minutes
  4. Heat the tubes for about 45 seconds in a 42°C water bath; do not shake
  5. Place on ice for 2 minutes
  6. Add 250 µl of room temperature SOC Medium to each tube
  7. Shake at 37°C (300 rpm) for more than 30 minutes.
  8. Light the bunsen burner
  9. Using two sets of petri dishes containing LB agar w/ Kanamycin (1 w/ 5 µl of cells and one w/ 50 µl of cells):
    1. 5 µl: pipette 5 µl of cells with 45 µl of SOC onto the agar mix
    2. 50 µl: pipette 50 µl of cells onto the agar mix.
  10. For each plate, we use sterile technique to spread the agar:
    1. Take the metal spreader and soak the bottom in ethanol, making sure to rinse exposed parts of the handle with distilled water.
    2. Very briefly put the ethanol soaked parts of the spreader under the flame.
    3. Touch the agar to make sure the spreader isn't too hot (will singe if too hot), and spread the cells around the plate.
  11. Afterwards, turn the plates upside down and incubate at 37°C

  • Day 2: Growing Bacteria in Liquid Medium (6/12/07)
  1. Take out the 6 grown colonies.
  2. Get 6 100 mL culture tubes.
  3. In each tube, use the pipet gun to add 2 mL of KB, and add 2 µl of Kanamycin (50 mg/mL)1000x
  4. Light the bunsen burner.
  5. Briefly place the tongs under the flame to sterilize, and use them to pick up a sterile toothpick.
  6. Use the toothpick to gather a single colony of cells, and place the toothpick and the colony into the solution.
  7. Repeat this sterile technique with the other 6.
  8. Shake at 37°C (300 rpm) for more than 30 minutes.
  9. Incubate overnight

  • Day 3: DNA Plasmid Preps
  1. Take out the 6 tubes of grown bacteria in liquid medium and spin two tubes with 1 mL in each for 1 minute at high speed.
  2. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
    1. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
    2. If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
  3. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4–6 times.
    1. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
    2. If LyseBlue has been added to Buffer P1 the cell suspension will turn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.
  4. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
    1. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to 10 times. The solution should become cloudy.
    2. If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.
  5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
    1. A compact white pellet will form.
  6. Apply the supernatants that resulted from step 4 to the QIAprep spin column by decanting or pipetting.
  7. Centrifuge for a minute. Discard the flow-through.
  8. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for a minute. Discard the flow-through.
    1. This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α™ do not require this additional wash step.
  9. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for a minute. Discard flow-through.
  10. Centrifuge for an additional 1 min to remove residual wash buffer.
    1. Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
  11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Protocol Recorded by Kevin Shee

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