Team:NTU-Singapore/Notebook/26 May 2008

From 2008.igem.org

(Difference between revisions)
(New page: =Monday,26 May= *For this whole week, we'll do the modeling tasks. ==Morning:== *List out all the chemicals for order. ==Afternoon:== *Start modeling.)
Line 5: Line 5:
==Afternoon:==
==Afternoon:==
*Start modeling.
*Start modeling.
 +
*[[User:Greenbear|Greenbear]] and Choon Kit: This afternoon we have done the extraction and transformation of 2 plasmids in the registry, ''iron promoter'' and ''LacI''. We follow the steps instructed by the Registry, which can be found [http://partsregistry.org/Help:Spring_2008_DNA_distribution Here]. Below I copied it for you guys to see. The only difference is that we only incubated the cells at 37ºC for ''1 hours'' instead of 2 while the tubes are rotating. We then left the cells for overnight incubation and tomorrow will make the glycerol stock. Any difficulty in understanding the steps, just refer to the filmed videos at our "main computer"
 +
<br><br>
 +
{|border="1" style="background-color:#ffffcc;"
 +
|
 +
===Locate the Part===
 +
 +
Use the Registry tools to locate the desired part.  It is important to note the plasmid and antibiotic resistance(s) of the required part.  See more information [http://partsregistry.org/Help:IGEM_08_DNA_distribution#Locating_a_Part_in_the_Distribution Here].
 +
 +
 +
===Paper Punches===
 +
<b>
 +
Estimated time: 1 minute per spot
 +
 +
Materials needed:
 +
*TE (10:1, pH 8.0)
 +
*Desired spot location information
 +
*Olfa cutting mat (back of binder)
 +
*Punch tool
 +
*0.5ml PCR tubes
 +
</b>
 +
 +
 +
# Warm up 5 μl aliquots of TE buffer to 50ºC in 0.5 ml (PCR) Eppendorf tubes.  Warm a water bath to 42 degrees.
 +
# Slide the cutting mat under the page in the binder containing the DNA of your part.
 +
# Using the punch tool, press down firmly on the spot you want to punch out while rotating the punch tool.  The spot is large enough to allow several punches, so punch at the edge of the spot.
 +
# Eject the punched paper spot into the tube containing 5 µL of TE buffer by pressing down on the top part of the punch tool.
 +
# Be sure to clean the punch tool before punching out another spot, to prevent cross-contamination of parts.
 +
 +
 +
 +
 +
===Punch Tool Cleaning===
 +
<b>
 +
Estimated time: 5 minutes per spot
 +
 +
Materials needed:
 +
*Blotting paper (back of binder)
 +
*10% bleach
 +
*diH2O
 +
*95% ethanol
 +
</b>
 +
# Punch a blank sheet of blotting paper (back of the notebook) and discard the punched paper. Pull the punch tool apart, so that the black rod is separated from the column.
 +
# Dip both the rod and the column briefly into a series of solutions of 10% bleach, distilled water, a second bath of distilled water, and a final bath of 95% ethanol.  We have found that strong detergents tend to remain on the punch tool and can inhibit transformation of the DNA.  Ethanol is preferred over isopropanol for the final cleaning bath, as it dries much faster.
 +
# Blot with a Kimwipe and allow to dry for 5 minutes.  Note that the ethanol can wick up inside the column of the punch tool.  It is important that the punch tool be completely dry before punching out another DNA spot, as the ethanol will affect the transformation efficiency.
 +
 +
 +
 +
===Transformation===
 +
<b>
 +
Estimated time: 3 hours (plus 12-14 hour incubation)
 +
 +
Materials needed:
 +
*Spots soaked in TE
 +
*2.0ml conical bottom tubes (one per spot)
 +
*Ice
 +
*Competent cells
 +
*42º water bath / 37º incubator
 +
*SOC (check for contamination!)
 +
*Petri plates with appropriate antibiotic
 +
</b>
 +
 +
# Soak the spots in 5 µL 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 labeled 2 ml conical bottom tubes on wet ice.  Add 2 µL of DNA in TE and  50 µL of thawed TOP10 competent cells to the tubes. In our experience, these volumes have the best transformation efficiency.  The 2 ml tubes allow better liquid movement during incubation.  Extra eluted DNA may be held at least several weeks frozen or at refrigerator temperature.
 +
# Hold the DNA and competent cells on ice for 30 minutes.  This improves transformation efficiency by a significant amount.
 +
# Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.  A water bath is important to improve heat transfer to the cells.
 +
# Incubate the cells on ice for 2 minutes.
 +
# Add 200 μl of SOC broth (check that this broth is not turbid, which would indicate previous contamination and bacterial growth).  This broth should contain no antibiotics.
 +
# Incubate the cells at 37ºC for '''2 hours''' while the tubes are rotating or shaking.  We have found that growth for 2 hours helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
 +
# Label an LB agar plate containing the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance.  Plate 250 µl of the incubated cell culture on the plate.
 +
# 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.
 +
|}

Revision as of 02:36, 27 May 2008

Contents

Monday,26 May

  • For this whole week, we'll do the modeling tasks.

Morning:

  • List out all the chemicals for order.

Afternoon:

  • Start modeling.
  • Greenbear and Choon Kit: This afternoon we have done the extraction and transformation of 2 plasmids in the registry, iron promoter and LacI. We follow the steps instructed by the Registry, which can be found [http://partsregistry.org/Help:Spring_2008_DNA_distribution Here]. Below I copied it for you guys to see. The only difference is that we only incubated the cells at 37ºC for 1 hours instead of 2 while the tubes are rotating. We then left the cells for overnight incubation and tomorrow will make the glycerol stock. Any difficulty in understanding the steps, just refer to the filmed videos at our "main computer"



Locate the Part

Use the Registry tools to locate the desired part. It is important to note the plasmid and antibiotic resistance(s) of the required part. See more information [http://partsregistry.org/Help:IGEM_08_DNA_distribution#Locating_a_Part_in_the_Distribution Here].


Paper Punches

Estimated time: 1 minute per spot

Materials needed:

  • TE (10:1, pH 8.0)
  • Desired spot location information
  • Olfa cutting mat (back of binder)
  • Punch tool
  • 0.5ml PCR tubes


  1. Warm up 5 μl aliquots of TE buffer to 50ºC in 0.5 ml (PCR) Eppendorf tubes. Warm a water bath to 42 degrees.
  2. Slide the cutting mat under the page in the binder containing the DNA of your part.
  3. Using the punch tool, press down firmly on the spot you want to punch out while rotating the punch tool. The spot is large enough to allow several punches, so punch at the edge of the spot.
  4. Eject the punched paper spot into the tube containing 5 µL of TE buffer by pressing down on the top part of the punch tool.
  5. Be sure to clean the punch tool before punching out another spot, to prevent cross-contamination of parts.



Punch Tool Cleaning

Estimated time: 5 minutes per spot

Materials needed:

  • Blotting paper (back of binder)
  • 10% bleach
  • diH2O
  • 95% ethanol

  1. Punch a blank sheet of blotting paper (back of the notebook) and discard the punched paper. Pull the punch tool apart, so that the black rod is separated from the column.
  2. Dip both the rod and the column briefly into a series of solutions of 10% bleach, distilled water, a second bath of distilled water, and a final bath of 95% ethanol. We have found that strong detergents tend to remain on the punch tool and can inhibit transformation of the DNA. Ethanol is preferred over isopropanol for the final cleaning bath, as it dries much faster.
  3. Blot with a Kimwipe and allow to dry for 5 minutes. Note that the ethanol can wick up inside the column of the punch tool. It is important that the punch tool be completely dry before punching out another DNA spot, as the ethanol will affect the transformation efficiency.


Transformation

Estimated time: 3 hours (plus 12-14 hour incubation)

Materials needed:

  • Spots soaked in TE
  • 2.0ml conical bottom tubes (one per spot)
  • Ice
  • Competent cells
  • 42º water bath / 37º incubator
  • SOC (check for contamination!)
  • Petri plates with appropriate antibiotic

  1. Soak the spots in 5 µL 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 labeled 2 ml conical bottom tubes on wet ice. Add 2 µL of DNA in TE and 50 µL of thawed TOP10 competent cells to the tubes. In our experience, these volumes have the best transformation efficiency. The 2 ml tubes allow better liquid movement during incubation. Extra eluted DNA may be held at least several weeks frozen or at refrigerator temperature.
  3. Hold the DNA and competent cells on ice for 30 minutes. This improves transformation efficiency by a significant amount.
  4. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath is important to improve heat transfer to the cells.
  5. Incubate the cells on ice for 2 minutes.
  6. Add 200 μl of SOC broth (check that this broth is not turbid, which would indicate previous contamination and bacterial growth). This broth should contain no antibiotics.
  7. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. We have found that growth for 2 hours helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
  8. Label an LB agar plate containing the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 250 µl of the incubated cell culture on the plate.
  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.
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