Team:Cornell/Week 6

From 2011.igem.org

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<font size="5"> <i> June 10th - June 16th </i></font>
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<font size="5"> <i> July 10th - July 16th </i></font>
</html><br><br>
</html><br><br>
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==Sunday==
+
==Sunday, July 10==
-
Lab work done by: James Mathew & Charlie Chung
+
Lab work done by: James Mathew, Charlie Chung
-
*Worked on Website.  Preliminary design of banner, safety content, and temporary lab notebook spreadsheet.
+
:*Worked on website
-
*Ligation product was transformed in electrocompetent cells, culture plated on Amp
+
::- Preliminary design of banner, safety content, and temporary lab notebook spreadsheet
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*Vio-Operon plasmid was transformed and plated on Kanamyacin
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:*Ligation product was transformed in electrocompetent cells
-
*RFP PCR done overnight
+
:*Culture plated on agar plate treated with ampicillin
 +
:*Plasmid containing the Vio operon was transformed and plated on agar treated with kanamycin
 +
:*Overnight PCR reaction for RFP
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==Monday==
+
==Monday, July 11==
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==Tuesday==
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==Tuesday, July 12==
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*Finished final design of team logo.
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:*Finished final design of team logo
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==Wednesday==
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==Wednesday, July 13==
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*Website Meeting to continue design and construction of website.  Designed and constructed final banner design.
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:*"Website Team" meeting to continue design and construction of website
 +
::- Designed and constructed final banner design
-
==Thursday==
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==Thursday, July 14==
-
Lab work done by: James Mathew & Charlie Chung
+
Lab work done by: James Mathew, Charlie Chung
-
*Sequencing revealed no GFP. Retry the 3-piece ligation of GFP + Annealed Primer Pair A (half of biotin tag) + Annealed Primer Pair B (other half of biotin tag & iGEM suffix).
+
:*Sequencing revealed no GFP in the ligation product
-
*PCR to amplify vector backbone containing RFP.
+
:*Will retry the 3-piece ligation of GFP + Annealed Primer Pair A (half of biotin tag) + Annealed Primer Pair B (other half of biotin tag & iGEM suffix)
-
*Met Prof. Lucks -- prospective iGEM adviser and coolest man alive
+
:*PCR to amplify vector backbone containing RFP
 +
:*Met Prof. Lucks -- prospective iGEM advisor and coolest man alive
-
==Friday==
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==Friday, July 15==
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*Banner was updated to include flash animation of moving gears.
+
:*Banner was updated to include Flash animation of moving gears.
'''Team Meeting'''
'''Team Meeting'''
-
#Ligation didn't work, need to retry it over the weekend (transform and plate today, innoculate and colony PCR saturday, miniprep and prepare for sequencing on sunday).
+
#Ligation didn't work -- retry over the weekend
-
#Prepare more culture of backbone plasmid (innoculate today, miniprep on saturday)
+
#::- transform and plate today
-
#We want to order primers today! for other methods of ligation, with longer primers, and two step PCR with short primers, in case our current one doesn't work. Also going to order primers for our pathway enzymes. The primers should come Wednesday. If ligation this weekend fails, we can try these other PCR methods.
+
#::- inoculate and colony PCR on Saturday
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#Animations and website are continuing
+
#::- Miniprep and prepare for sequencing on Sunday
-
Lab work done by: James Matthews, Charlie Chung & Youjin Cho
+
#Prepare more culture of backbone plasmid
-
*Running low on pZE vector backbone, so borrowed two culture tubes from Sean. Miniprep to purify out the plasmid. Followed standard Qiagen Miniprep protocol for microcentrifuge.
+
#::- inoculate today
-
*Verify if PCR amplification of RFP worked using gel electrophoresis. If yes, then proceed to gel extraction and purification. NOTE: thermocycler did not keep our rxn tube @ 4 deg C
+
#::- Miniprep on Saturday
-
*Electroporation method of transforming our 3-piece ligation product and control into DH5α (competent E. coli = made porous for uptake of outside DNA). Plated transformed bacteria onto LB + Ampicillin. Followed protocol as written in the "Multimedia" section.
+
#Order primers today for other methods of ligation: (1) longer primers (2) two-step PCR with shorter primers
 +
#Order primers for our pathway enzymes: VioA, VioB, VioB
 +
#:- Primers should come Wednesday
 +
#:- If ligation this weekend fails, we can try these other PCR methods
 +
#Animations and website construction are continuing
 +
Lab work done by: James Mathew, Charlie Chung, Youjin Cho
 +
:*Running low on pZE vector backbone, so fired up two cultures
 +
:*Miniprep to purify out the plasmid. Followed standard Qiagen Miniprep protocol for microcentrifuge
 +
:*Verify if PCR amplification of RFP worked using gel electrophoresis. If yes, then proceed to gel extraction and purification
 +
::- ''NOTE: Thermocycler did not keep our rxn tube at 4°C''
 +
:*Electroporation method of transforming our 3-piece ligation product and control into DH5α (competent E. coli = made porous for uptake of outside DNA)
 +
:*Plated transformed bacteria onto agar plates treated with ampicillin
-
==Saturday==
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==Saturday, July 16==
-
Lab work done by: Youjin Cho and Nicholas Kramer
+
Lab work done by: Youjin Cho, Nicholas Kramer
-
*Looked at the plates for ligation. Ligation worked!! Sean will pick 2 colonies off the plate in the evening and innoculate overnight.
+
:*Growth of numerous colonies suggests the ligation reaction worked
-
*PCR to amplify vector backbone containing RFP.
+
::- Will pick 2 colonies off the plate in the evening and inoculate overnight
-
**Melting temperature of 57C leads to non-specific binding. We ran 2 PCR reactions with melting temperatures 59C and 61C.
+
:*PCR to amplify vector backbone containing RFP
-
__NOTOC__
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::- Melting temperature of 57°C leads to non-specific binding
 +
::- Ran 2 PCR reactions with melting temperatures 59°C and 61°C

Latest revision as of 17:22, 18 September 2011

Results | Protocol | Notebook | Parts Submitted

Week 1 | Week 2 - 5 | Week 6 | Week 7 | Week 8 | Week 9 | Week 10 | Week 11 | Week 12 | Week 13 | Week 14 | Week 15 | Week 16 | Week 17 | Week 18 | Week 19 | Week 20 | Week 21 |


July 10th - July 16th

Sunday, July 10

Lab work done by: James Mathew, Charlie Chung

  • Worked on website
- Preliminary design of banner, safety content, and temporary lab notebook spreadsheet
  • Ligation product was transformed in electrocompetent cells
  • Culture plated on agar plate treated with ampicillin
  • Plasmid containing the Vio operon was transformed and plated on agar treated with kanamycin
  • Overnight PCR reaction for RFP

Monday, July 11

Tuesday, July 12

  • Finished final design of team logo

Wednesday, July 13

  • "Website Team" meeting to continue design and construction of website
- Designed and constructed final banner design

Thursday, July 14

Lab work done by: James Mathew, Charlie Chung

  • Sequencing revealed no GFP in the ligation product
  • Will retry the 3-piece ligation of GFP + Annealed Primer Pair A (half of biotin tag) + Annealed Primer Pair B (other half of biotin tag & iGEM suffix)
  • PCR to amplify vector backbone containing RFP
  • Met Prof. Lucks -- prospective iGEM advisor and coolest man alive

Friday, July 15

  • Banner was updated to include Flash animation of moving gears.

Team Meeting

  1. Ligation didn't work -- retry over the weekend
    - transform and plate today
    - inoculate and colony PCR on Saturday
    - Miniprep and prepare for sequencing on Sunday
  2. Prepare more culture of backbone plasmid
    - inoculate today
    - Miniprep on Saturday
  3. Order primers today for other methods of ligation: (1) longer primers (2) two-step PCR with shorter primers
  4. Order primers for our pathway enzymes: VioA, VioB, VioB
    - Primers should come Wednesday
    - If ligation this weekend fails, we can try these other PCR methods
  5. Animations and website construction are continuing

Lab work done by: James Mathew, Charlie Chung, Youjin Cho

  • Running low on pZE vector backbone, so fired up two cultures
  • Miniprep to purify out the plasmid. Followed standard Qiagen Miniprep protocol for microcentrifuge
  • Verify if PCR amplification of RFP worked using gel electrophoresis. If yes, then proceed to gel extraction and purification
- NOTE: Thermocycler did not keep our rxn tube at 4°C
  • Electroporation method of transforming our 3-piece ligation product and control into DH5α (competent E. coli = made porous for uptake of outside DNA)
  • Plated transformed bacteria onto agar plates treated with ampicillin

Saturday, July 16

Lab work done by: Youjin Cho, Nicholas Kramer

  • Growth of numerous colonies suggests the ligation reaction worked
- Will pick 2 colonies off the plate in the evening and inoculate overnight
  • PCR to amplify vector backbone containing RFP
- Melting temperature of 57°C leads to non-specific binding
- Ran 2 PCR reactions with melting temperatures 59°C and 61°C
Retrieved from "http://2011.igem.org/Team:Cornell/Week_6"