Team:Calgary/7 July 2010

From 2010.igem.org

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{{CalgaryNotebookTemplate|
{{CalgaryNotebookTemplate|
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'''Wednesday July 7, 2010'''
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Wednesday July 7, 2010|
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[[Image:07.07.2010. I0500+B0034 and E0040+B0015.jpg|thumb|400px|Wells 2-9 Jeremy's I0500+B0034 with and without restriction enzymes. Wells 11-19 Dev's E0040+B0015 with and without enzymes.]]
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<u>Emily</u>
 +
 
 +
*Today I finalized the enzymes that we are going to be using for the multiple cloning sites in our transcription/ translation testing circuit.  I also had an ethics meeting with Dev and Raida.  We talked about what previous iGEM teams have done in terms of Ethics as well as what we are going to be presenting at Suffield next week.  I strated reading a few papers on snythetic biology ethics to come up with soe quetsions for next week.  Today I also made ovenright cultures of Himika's E1010-B0015 construct as well as her E1010 plasmid switch.
<u>Raida</u>
<u>Raida</u>
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*Today I completed the transformation of my construct (R0040-I0504) and plated them in 6 different Ampicilin Resistant plates.
+
*Today I completed the transformation of my construct (R0040-I3504) and plated them in 6 different Ampicilin Resistant plates.
'''Procedure: Transformation'''
'''Procedure: Transformation'''
-
*1. Thaw Competent Cells
+
# Thaw Competent Cells
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*2. Add 20 uL DNA
+
# Add 20 uL DNA
-
*3. Ice for 1/2 hour, Heat Shock 5 minutes at 37 degrees Celcius, Ice 5 min.
+
# Ice for 1/2 hour, Heat Shock 5 minutes at 37 degrees Celcius, Ice 5 min.
-
*4. Recover with 250 uL SOC for 30 minutes
+
# Recover with 250 uL SOC for 30 minutes
-
*5. Spin down at 14 RPM, Concentrate to approximately 100 uL
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# Spin down at 14 RPM, Concentrate to approximately 100 uL
-
*6. Plate 25 uL on plate 1,2,3 and Plate 50 uL on plate 4,5,6
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# Plate 25 uL on plate 1,2,3 and Plate 50 uL on plate 4,5,6
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*7. Leave in Incubator overnight for 20 hours
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# Leave in Incubator overnight for 20 hours
'''The plates are labeled as following:'''
'''The plates are labeled as following:'''
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Today I completed the plasmid switch for I0500 into pSB1AK3, mini prep I0500+B0034 with Sigma-Aldrich and finished a restriction digest of EcoRI and PstI on I0500+B0034
Today I completed the plasmid switch for I0500 into pSB1AK3, mini prep I0500+B0034 with Sigma-Aldrich and finished a restriction digest of EcoRI and PstI on I0500+B0034
-
 
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*'''At the end of the heat killing process:'''
 
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5 uL Antartic Phosphotase Buffer
 
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5 uL of Water and
 
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1 uL Antartic Phosphotase
 
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It was placed in the water bath at 37°C for half an hour and then I ligated the insert and the vector using
 
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5 uL of Insert and 5 uL of Vector
 
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1 uL Quick Ligase
 
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10 uL 2x Quick Ligase Buffer
 
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The construct is left in the fridge overnight and transformation will be done tomorrow.
 
-
 
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*''see protocol for Gen-Elute Mini Prep Sigma Aldrich Kit''
 
-
 
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*'''Restriction Digest'''- This is the I0500+B0034 (same for all 4 colonies)
 
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**-2 uL DNA
 
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**-0.5uL EcoRI
 
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**-0.5uL PstI
 
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**-3.5uL RE2 Buffer
 
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**-8.5uL H2O
 
*37 degrees for 1 hour and heat kill 80 degrees for 20 minutes
*37 degrees for 1 hour and heat kill 80 degrees for 20 minutes
*ran on 1% agarose gel at 90V
*ran on 1% agarose gel at 90V
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<u>Chris</u>
<u>Chris</u>
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Today, I drafted the potential budget for this year's project. The total expenses this year are projected to amount to $64 500 with that money being used towards paying students, travelling and lodging at MIT, paying for laboratory materials, and buying T-shirts as well as a mascot suit. Our current financial status is sufficient to cover approximately 80% of this with $54 600 in total funds. These have been provided by BioAlberta, Alberta Innovates Technology Funds, O'Brien Student Centre, and Alberta Heritage Foundation for Medical Research.  
Today, I drafted the potential budget for this year's project. The total expenses this year are projected to amount to $64 500 with that money being used towards paying students, travelling and lodging at MIT, paying for laboratory materials, and buying T-shirts as well as a mascot suit. Our current financial status is sufficient to cover approximately 80% of this with $54 600 in total funds. These have been provided by BioAlberta, Alberta Innovates Technology Funds, O'Brien Student Centre, and Alberta Heritage Foundation for Medical Research.  
-
As well, in the morning, Dev and I used the Sigma Aldrich GenElute Plasmid Preparation kit and modified instructions to isolate the plasmids of the constructions of I0500 - B0034 and E0040-B0015 as well as the parts of I0500, I13507, and E1010. The procedure used was as follows:
+
As well, in the morning, Dev and I used the Sigma Aldrich GenElute Plasmid Preparation kit and modified instructions to isolate the plasmids of the constructions of I0500 - B0034 and E0040-B0015 as well as the parts of I0500, I13507, and E1010.  
-
 
+
-
#Pellet the cells from the overnight cultures using a centrifuge for 20 minutes at a speed of 4000 rpm at 4&deg;C.
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#Discard the supernatant, while being careful not to remove any of the pellet
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#Resuspend the pellet in 200 μL of Resuspension Solution (with RNase A added)
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#Transfer the solution from a Falcon tube to a properly labelled 1.5 μL microcentrifuge tube.
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#Add 200 μL of the Lysis solution and invert gently to mix. Allow the mixture to clear for 4.5 minutes.
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#Add 350 μL of Neutralization Solution and invert the tube 4-6 times to mix.
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#Pellet the microcentrifuge tubes at 14 000 rpm using a microcentrifuge for 15 minutes. The solution will be known as the lysate.
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#Add 500 μL of the Column Preparation Solution to a binding column inside a collection tube. Centrifuge this tube for 1 minute at 14 000 rpm and discard the resulting liquid under the binding tube.
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#Transfer the lysate from the microcentrifuge tube into the binding column while being careful not to transfer any solid. The microcentrifuge tube may now be discarded.
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#Centrifuge the collection tubes at 14 000 rpm for 1 minute and discard the liquid that flowed through the binding column.
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#Add 750 μL of Wash Solution with concentrated ethanol added to the column and centrifuge at 14 000 rpm for 1 minute. Discard the liquid that flowed through into the collection tube.
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#Repeat this procedure a second time with Wash Solution.
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#Spin the tube for 1 minute at 14 000 rpm to dry the column.
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#Transfer the column into a properly labelled 1.5 μL microcentrifuge tube.
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#Add 50 μL of double-distilled water to the column and spin for 1 minute at 14 000 rpm.
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#Use a spectrophotometer to measure the concentration and purity of the plasmids.
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In the afternoon, I contacted a General Manager from New England Biolabs about possible sponsorship and received a positive response. They will send some enzymes to us in the coming week.  
In the afternoon, I contacted a General Manager from New England Biolabs about possible sponsorship and received a positive response. They will send some enzymes to us in the coming week.  
 +
 +
 +
<u>Alex</u>
 +
 +
The overnight colonies of the cpxp in pSB1AK3 was pink, which means it was expressing RFP and the construction was unsuccessful. We have decided it would be best to wait for the new primer to come so that we can create new PCR product and construct with that because it contains the entire biobrick suffix, which eliminates the risk of scarring. Today I prepared slides for a presentation to our supervisors on Friday, also I attempted to get into contact with my local newspaper in Bow Island to see if they will do a story on iGEM, no response yet but I’m confident we can get in.
 +
 +
 +
<u>Patrick</u>
 +
 +
The overnight colonies were pink, save for a single colony. This suggests that RFP was produced, meaning the plasmid switch was unsuccessful. Upon checking the original plates, even the colony that did not turn pink in the culture was pink. Baffling, no doubt, but the conclusion is clear: continuing to use ''XbaI'' and ''SpeI'' as plasmid insertion sites will definitely be much more trouble than it is worth. Therefore, we ordered in a new reverse primer that also contained the ''NotI'' and ''PstI'' sites. This will hopefully make the plasmid switch directional.
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Latest revision as of 03:36, 23 August 2010

Notebook

May
MTWTFSS
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31

June
MTWTFSS
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30

July
MTWTFSS
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31

August
MTWTFSS
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31

September
MTWTFSS
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30

October
MTWTFSS
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

Wednesday July 7, 2010

Wells 2-9 Jeremy's I0500+B0034 with and without restriction enzymes. Wells 11-19 Dev's E0040+B0015 with and without enzymes.

Emily

  • Today I finalized the enzymes that we are going to be using for the multiple cloning sites in our transcription/ translation testing circuit. I also had an ethics meeting with Dev and Raida. We talked about what previous iGEM teams have done in terms of Ethics as well as what we are going to be presenting at Suffield next week. I strated reading a few papers on snythetic biology ethics to come up with soe quetsions for next week. Today I also made ovenright cultures of Himika's E1010-B0015 construct as well as her E1010 plasmid switch.


Raida

  • Today I completed the transformation of my construct (R0040-I3504) and plated them in 6 different Ampicilin Resistant plates.

Procedure: Transformation

  1. Thaw Competent Cells
  2. Add 20 uL DNA
  3. Ice for 1/2 hour, Heat Shock 5 minutes at 37 degrees Celcius, Ice 5 min.
  4. Recover with 250 uL SOC for 30 minutes
  5. Spin down at 14 RPM, Concentrate to approximately 100 uL
  6. Plate 25 uL on plate 1,2,3 and Plate 50 uL on plate 4,5,6
  7. Leave in Incubator overnight for 20 hours

The plates are labeled as following:

  • P1: R0040-I13504 (construct tube C 6 July) 25 uL
  • P2: R0040-I13504 (construct tube D 6 July) 25 uL
  • P3: R0040-I13504 (construct tube F 6 July) 25 uL
  • P4: R0040-I13504 (construct tube C 6 July) 50 uL
  • P5: R0040-I13504 (construct tube D 6 July) 50 uL
  • P6: R0040-I13504 (construct tube F 6 July) 50 uL

Then I left the plates in the Incubator which is at 37 degrees Celcius. I will leave it there until 10:00 am tomorrow (July 8th), which would let the cells grow for 20 hours.


Jeremy

Today I completed the plasmid switch for I0500 into pSB1AK3, mini prep I0500+B0034 with Sigma-Aldrich and finished a restriction digest of EcoRI and PstI on I0500+B0034

  • 37 degrees for 1 hour and heat kill 80 degrees for 20 minutes
  • ran on 1% agarose gel at 90V


Chris

Today, I drafted the potential budget for this year's project. The total expenses this year are projected to amount to $64 500 with that money being used towards paying students, travelling and lodging at MIT, paying for laboratory materials, and buying T-shirts as well as a mascot suit. Our current financial status is sufficient to cover approximately 80% of this with $54 600 in total funds. These have been provided by BioAlberta, Alberta Innovates Technology Funds, O'Brien Student Centre, and Alberta Heritage Foundation for Medical Research.

As well, in the morning, Dev and I used the Sigma Aldrich GenElute Plasmid Preparation kit and modified instructions to isolate the plasmids of the constructions of I0500 - B0034 and E0040-B0015 as well as the parts of I0500, I13507, and E1010.

In the afternoon, I contacted a General Manager from New England Biolabs about possible sponsorship and received a positive response. They will send some enzymes to us in the coming week.


Alex

The overnight colonies of the cpxp in pSB1AK3 was pink, which means it was expressing RFP and the construction was unsuccessful. We have decided it would be best to wait for the new primer to come so that we can create new PCR product and construct with that because it contains the entire biobrick suffix, which eliminates the risk of scarring. Today I prepared slides for a presentation to our supervisors on Friday, also I attempted to get into contact with my local newspaper in Bow Island to see if they will do a story on iGEM, no response yet but I’m confident we can get in.


Patrick

The overnight colonies were pink, save for a single colony. This suggests that RFP was produced, meaning the plasmid switch was unsuccessful. Upon checking the original plates, even the colony that did not turn pink in the culture was pink. Baffling, no doubt, but the conclusion is clear: continuing to use XbaI and SpeI as plasmid insertion sites will definitely be much more trouble than it is worth. Therefore, we ordered in a new reverse primer that also contained the NotI and PstI sites. This will hopefully make the plasmid switch directional.

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