Team:NTU-Singapore/Notebook/26 May 2008

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=Monday,26 May=
=Monday,26 May=
*For this whole week, we'll do the modeling tasks.
*For this whole week, we'll do the modeling tasks.
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==Afternoon:==
==Afternoon:==
*Start modeling.
*Start modeling.
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*[[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"  
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*[[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].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"  
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{|border="1" style="background-color:#ffffcc;" cellpadding="20"
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===Locate the Part===
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==Experiment No 1==
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  Date 26 May 2008
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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].
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  Start Time 1500
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  End Time 1730
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===Paper Punches===
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<b>
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Estimated time: 1 minute per spot
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Materials needed:
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*TE (10:1, pH 8.0)
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*Desired spot location information
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*Olfa cutting mat (back of binder)
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*Punch tool
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*0.5ml PCR tubes
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</b>
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# Warm up 5 μl aliquots of TE buffer to 50ºC in 0.5 ml (PCR) Eppendorf tubes. Warm a water bath to 42 degrees.
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# Slide the cutting mat under the page in the binder containing the DNA of your part.
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# 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.
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# 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.
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# Be sure to clean the punch tool before punching out another spot, to prevent cross-contamination of parts.
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===Punch Tool Cleaning===
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<b>
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Estimated time: 5 minutes per spot
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Materials needed:
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===Personnel:===
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*Blotting paper (back of binder)
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Choon Kit, Hung, Dr Tan TL <br>
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*10% bleach
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*diH2O
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*95% ethanol
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</b>
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# 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.
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# 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.
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# 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.
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===Title of Experiment:===
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Experiment could be classified in 3 phases:
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I. Preparation for Punch Tool Cleaning
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II. Paper Punching of  Biobrick
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III. Transformation
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<br>
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===Materials:===
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''Phase I:''
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8 x 2.0ml conical bottom tubes
 +
3 x weighting tray
 +
Kimwipes
 +
Blotting paper (back of binder)
 +
10% bleach (chlorine)
 +
DI water
 +
95% ethanol
 +
Pippette with pipette tips
 +
''Phase II:''
 +
TE (10:1, pH 8.0)
 +
Olfa Cutting Mat (back of binder)
 +
Punch Tool
 +
0.5ml PCR Tubes
 +
Pippette and Pipette Tips
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===Transformation===
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''Phase III:''
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<b>
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Spots soaked in TE
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Estimated time: 3 hours (plus 12-14 hour incubation)
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2.0 ml conical bottom tubes (one per spot)
 +
Ice
 +
Competent cells
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42oC water bath / 37oC incubator
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LB or SOC broth
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Agar Plates with appropriate antibiotic (Ampicillin in this case)
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Transformation control DNA (e.g.  PUC18 plasmid)
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Materials needed:
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===Protocols/Procedures:===
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*Spots soaked in TE
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====Phase I:====
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*2.0ml conical bottom tubes (one per spot)
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#To 8 conical bottom tubes, 3 of them filled with 1ml of 10% bleach each, 3 filled with 1ml of DI water, and the last 2 filled with 1 ml of 95% ethanol.
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*Ice
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#Practice punching using blotting paper before beginning experiment.
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*Competent cells
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#When after each punch of plasmid, the puncher is washed by tipping into the contents of the 8 conical tubes in  a weighing tray in the following sequence: 3 times bleach, 3 times DI water, 2 times ethanol.
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*42º water bath / 37º incubator
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#Blot with a Kimwipe (high grade tissue) and allow to dry for 5 minutes before punching another spot of plasmid.
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*SOC (check for contamination!)
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====Phase II:====
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*Petri plates with appropriate antibiotic
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#Pipette 5ul of TE buffer to 50oC in PCR Eppendorf tubes. Warm a water bath to 42oC
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</b>
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#Using the cutting mat as a backing, a sample of plasmid is punched off the edge of the spot using the puncher by pressing firmly down and rotating the punch tool. (Do not press the ejection button)
 +
#Eject the punched paper spot into the PCR tube by pressing the ejection button
 +
#Clean punch tool to prevent cross contamination as mentioned in phase I
 +
====Phase III:====
 +
#Soak the spots in 5ul of warmed TE for 20 minutes. Also, thaw the competent cells on wet crushed ice.
 +
#Chill labeled 2ml concial bottom tubes on wet ice. Add 2ul of DNA in TE and 50ulof thawed competent cells to the tubes. Extra eluted DNA can be kept frozen at -80oC for several weeks.
 +
#Also, do a control transformation using pUC18 plasmid DNA.
 +
#Hold the DNA and competent cells on ice for 30 minutes to increase transformation efficiency.
 +
#Heat shock the cells by immersion in a pre-heated water bath at 42oC for 1 minute. This is to improve heat transfer to the cells.
 +
#Incubate the whole floating tray of conical tubes on ice for 2 minutes.
 +
#Add 200ul of LB broth. (no antibiotic, check that broth is not turbid, which indicates contamination and bacterial growth)
 +
#Incubate the cells at 37oC for 1-2 hours. 2 hours is optimal for plasmids with other antibiotic resistance other than amipicillin.
 +
#Label an LB agar plate with amipicillin with the date, project name, and plasmid e.g. LBA – 26May08 – iGEM – Fe promoter, LBA – 26May08 – iGEM – LacI, LBA – 26May08 – iGEM – pUC18.
 +
#Plate 250ul of the incubated cell culture on the plate
 +
#Incubate the plate at 37oC overnight or 12-14 hours. Do not incubate too long as the antibiotic may break down, resulting in growth of untransformed cells.
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# 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.
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===Observations:===
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# 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.
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2 colonies observed in LacI LB plate, 0 colony in Fe2+ LB plate, positive control pUC-18 showed many colonies.  
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# Hold the DNA and competent cells on ice for 30 minutes.  This improves transformation efficiency by a significant amount.
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===Conclusion:===
-
# 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.
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Fe2+ not successfully transformed in home-made competent cells. LacI and pUC-18 transformation were successful.
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# Incubate the cells on ice for 2 minutes.
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===Notes:===
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# 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.
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Refer to video clip 26 May 08 for details.
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# 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.
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===To be follow up:===
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# 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.
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1. Repeat whole transformation of Fe2+ plasmid using commercially competent cells.<br>
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# 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.
+
2. Pick out 2 colonies from LacI LB plate into LB broth with ampicillin overnight.   
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Latest revision as of 00:25, 28 October 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].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"



Experiment No 1

Date 26 May 2008 
Start Time 1500						
End Time 1730

Personnel:

Choon Kit, Hung, Dr Tan TL

Title of Experiment:

Experiment could be classified in 3 phases: 

I.	Preparation for Punch Tool Cleaning
II.	Paper Punching of  Biobrick
III.	Transformation


Materials:

Phase I:
8 x 2.0ml conical bottom tubes
3 x weighting tray
Kimwipes 
Blotting paper (back of binder)
10% bleach (chlorine)
DI water
95% ethanol
Pippette with pipette tips

Phase II:
TE (10:1, pH 8.0)
Olfa Cutting Mat (back of binder)
Punch Tool
0.5ml PCR Tubes
Pippette and Pipette Tips

Phase III:
Spots soaked in TE
2.0 ml conical bottom tubes (one per spot)
Ice
Competent cells
42oC water bath / 37oC incubator
LB or SOC broth
Agar Plates with appropriate antibiotic (Ampicillin in this case)
Transformation control DNA (e.g.  PUC18 plasmid)

Protocols/Procedures:

Phase I:

  1. To 8 conical bottom tubes, 3 of them filled with 1ml of 10% bleach each, 3 filled with 1ml of DI water, and the last 2 filled with 1 ml of 95% ethanol.
  2. Practice punching using blotting paper before beginning experiment.
  3. When after each punch of plasmid, the puncher is washed by tipping into the contents of the 8 conical tubes in a weighing tray in the following sequence: 3 times bleach, 3 times DI water, 2 times ethanol.
  4. Blot with a Kimwipe (high grade tissue) and allow to dry for 5 minutes before punching another spot of plasmid.

Phase II:

  1. Pipette 5ul of TE buffer to 50oC in PCR Eppendorf tubes. Warm a water bath to 42oC
  2. Using the cutting mat as a backing, a sample of plasmid is punched off the edge of the spot using the puncher by pressing firmly down and rotating the punch tool. (Do not press the ejection button)
  3. Eject the punched paper spot into the PCR tube by pressing the ejection button
  4. Clean punch tool to prevent cross contamination as mentioned in phase I

Phase III:

  1. Soak the spots in 5ul of warmed TE for 20 minutes. Also, thaw the competent cells on wet crushed ice.
  2. Chill labeled 2ml concial bottom tubes on wet ice. Add 2ul of DNA in TE and 50ulof thawed competent cells to the tubes. Extra eluted DNA can be kept frozen at -80oC for several weeks.
  3. Also, do a control transformation using pUC18 plasmid DNA.
  4. Hold the DNA and competent cells on ice for 30 minutes to increase transformation efficiency.
  5. Heat shock the cells by immersion in a pre-heated water bath at 42oC for 1 minute. This is to improve heat transfer to the cells.
  6. Incubate the whole floating tray of conical tubes on ice for 2 minutes.
  7. Add 200ul of LB broth. (no antibiotic, check that broth is not turbid, which indicates contamination and bacterial growth)
  8. Incubate the cells at 37oC for 1-2 hours. 2 hours is optimal for plasmids with other antibiotic resistance other than amipicillin.
  9. Label an LB agar plate with amipicillin with the date, project name, and plasmid e.g. LBA – 26May08 – iGEM – Fe promoter, LBA – 26May08 – iGEM – LacI, LBA – 26May08 – iGEM – pUC18.
  10. Plate 250ul of the incubated cell culture on the plate
  11. Incubate the plate at 37oC overnight or 12-14 hours. Do not incubate too long as the antibiotic may break down, resulting in growth of untransformed cells.

Observations:

2 colonies observed in LacI LB plate, 0 colony in Fe2+ LB plate, positive control pUC-18 showed many colonies.

Conclusion:

Fe2+ not successfully transformed in home-made competent cells. LacI and pUC-18 transformation were successful.

Notes:

Refer to video clip 26 May 08 for details.

To be follow up:

1. Repeat whole transformation of Fe2+ plasmid using commercially competent cells.
2. Pick out 2 colonies from LacI LB plate into LB broth with ampicillin overnight.

Retrieved from "http://2008.igem.org/Team:NTU-Singapore/Notebook/26_May_2008"