Team:Wageningen UR/Journal/week10

From 2012.igem.org

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= week 10: 2 july - 6 july =
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= week 10: 2 july - 8 july =
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== Office work ==
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'''TuYV'''
'''TuYV'''
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A new transformation of last weeks ligation mixes into our electrocompetent DH5 Alfa ''E. Coli'' was performed. Results were unsatisfying.
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Monday:
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* A new transformation of last week's ligation mixes into our electro-competent DH5 Alfa ''E. Coli'' was performed. The next day we checked the colony, there were many colonies on the positive control, however there were the same level of colonies on the sample and negative control. The transformation was contaminated.  
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Two new gene variants were PCR amplified from the TuYV genome, adding iGEM pre- and suffixes and in one case a Histidine tag. Succesful results were acquired after three attempts.
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* Because there are iGEM illegal sites in our TuYV readthrough part, we designed a new construct named 4 and 4H, which were coat protein with part of the readthrough without illegal site and coat protein with part of the readthrough without illegal site with his-tag.  
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* We tried to isolate and amplify 4 and 4H today, but we did not get any band on the agrose gel check
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Tuesday:
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* Today we successfully PCR amplified the construct 4, which should be around 800bp.
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[[File:PCR amplification of 4.jpg|frame|center|Figure 1: Isolation of TuYV construct 4 from the TuYV genome]]
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Wednesday:
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* Today we tried another time to PCR amplify the construct 4H and we got expected band on the gel check.
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[[File:PCR amplification of 4H.jpg|frame|center|Figure 2: Isolation of TuYV construct 4H from the TuYV genome]]
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Written by: Wouter
 
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'''DLS boundary experiment'''
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''6th July'' (Mark)
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<ul>
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<li>Growing CCMV for the temperature and pH stability experiment</li>
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<li>Inoculated 200 mL of LB with BL21 that contains the IPTG inducible CCMV wild type monomer</li>
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<li>Followed protocol of producing CCMV</li>
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</ul>
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''7th – 10th July'' (Mark)
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<ul>
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<li>Dialysis CCMV VLPs</li>
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<li>Now running 1 times 50 mL dialysis tubes with CCMV wild type</li>
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<li>Followed protocol of dialysis of CCMV</li>
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</ul>
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----
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'''FPLC S400 preparations'''
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''2nd – 5th July'' (Mark)
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<ul>
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<li>Pouring and running of the S400 FPLC with the help of Serve Kengen.</li>
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</ul>
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''6th July'' (Mark)
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<ul>
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<li>FPLC with the S400 column</li>
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<li>1 run with 0.5 ml sample</li>
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<li>A very good separation with low concentration</li>
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</ul>
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[[https://2012.igem.org/Team:Wageningen_UR/Journal/week9 previous week]]          [[https://2012.igem.org/Team:Wageningen_UR/Journal/week11 next week]]
[[https://2012.igem.org/Team:Wageningen_UR/Journal/week9 previous week]]          [[https://2012.igem.org/Team:Wageningen_UR/Journal/week11 next week]]

Latest revision as of 22:59, 26 September 2012

week 10: 2 july - 8 july

Lab work

TuYV

Monday:

  • A new transformation of last week's ligation mixes into our electro-competent DH5 Alfa E. Coli was performed. The next day we checked the colony, there were many colonies on the positive control, however there were the same level of colonies on the sample and negative control. The transformation was contaminated.
  • Because there are iGEM illegal sites in our TuYV readthrough part, we designed a new construct named 4 and 4H, which were coat protein with part of the readthrough without illegal site and coat protein with part of the readthrough without illegal site with his-tag.
  • We tried to isolate and amplify 4 and 4H today, but we did not get any band on the agrose gel check

Tuesday:

  • Today we successfully PCR amplified the construct 4, which should be around 800bp.
Figure 1: Isolation of TuYV construct 4 from the TuYV genome

Wednesday:

  • Today we tried another time to PCR amplify the construct 4H and we got expected band on the gel check.
Figure 2: Isolation of TuYV construct 4H from the TuYV genome


DLS boundary experiment


6th July (Mark)

  • Growing CCMV for the temperature and pH stability experiment
  • Inoculated 200 mL of LB with BL21 that contains the IPTG inducible CCMV wild type monomer
  • Followed protocol of producing CCMV


7th – 10th July (Mark)

  • Dialysis CCMV VLPs
  • Now running 1 times 50 mL dialysis tubes with CCMV wild type
  • Followed protocol of dialysis of CCMV

FPLC S400 preparations


2nd – 5th July (Mark)

  • Pouring and running of the S400 FPLC with the help of Serve Kengen.


6th July (Mark)

  • FPLC with the S400 column
  • 1 run with 0.5 ml sample
  • A very good separation with low concentration

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