Team:Wageningen UR/Journal/week20

From 2012.igem.org

(Difference between revisions)
(→week 20: 10 September - 16 September)
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= week 20: 10 September - 16 September =
= week 20: 10 September - 16 September =
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'''TuYV'''
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'''Hepatitis B general'''
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Another two colonies PCR with pJET sequencing primers were done to find out colonise containing TuYV coat protein with  readthrough and coat protein with readthrough with his-tag. Finally another colony containing either the coat protein and readthrough or coat protein and readthrough with his-tag was identified.
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''10 September''
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All the colonies for colony PCR were minipreped.  
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* 2nd try colony PCR of the transformations from 4. September (with sequencing primers)
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TuYV constructs (PCR products) were digested, ligated into backbone from BBa_J04450 and transformed into MachI strain. In parallel, TuYV constructs (PCR products) were digested, ligated after the IPTG induced promoter again,and transformed into MachI.
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-> 6 out of 9 samples show an insert of the right size (see picture ''Colony PCR 12 September'')
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The next day we saw very promising colonies and the colony PCR confirmed that we successfully put TuYV insert into the chloramphenical backbone, but for adding them at the downstream of IPTG induced promoter, colony PCR revealed that there was only the backbone containing the IPTG induced promoter in the E.coli.
 
 +
''12 September''
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'''Polero'''
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* Miniprep and digestion check of colonies 5.Sept. (Hephis tag in BBa_J04450)
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Cells containing the plasmid( IPTG induced promoter with polero coat protein) were induced and lysised by French Press.
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-> Restriction check shows inserts that might be too large (expected size: around 600bp)
 +
(see picture ''restriction check 12 September'')
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Dialysis and purification were done. Samples were ready to send for EM test.
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* Sent samples for sequencing (HepB core protein + his tag in BBa_J04450)
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'''Hepatitis B general'''
 
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----
 
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''10 September''
 
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* 2nd try colony PCR of the transformations from 4. September (with sequencing primers)
 
-
-> 6 out of 9 samples show an insert of the right size
 
'''Hepatitis B outside modification'''
'''Hepatitis B outside modification'''
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* 2nd try colony PCR of the transformations from 4. September (with sequencing primers)
* 2nd try colony PCR of the transformations from 4. September (with sequencing primers)
 +
-> one colony seems to have the correct insert in the pSB1C3 backbone
-> one colony seems to have the correct insert in the pSB1C3 backbone
-> no conclusion can be made about samples containing the Hep-inside coil fragment in the Bba_J04500 brick  
-> no conclusion can be made about samples containing the Hep-inside coil fragment in the Bba_J04500 brick  
 +
(see picture ''Colony PCR 12 September'')
 +
''12 September''
 +
 +
* Miniprep and digestion check of colonies 5.Sept. (Hepinsidecoil in BBa_J04450)
 +
 +
-> Restriction check shows inserts that might be too large (expected size: around 600bp)
 +
 +
[[File:Restriction check 12.Sept.png]]
 +
 +
''restriction check 12 September''
 +
 +
 +
* Sent samples for sequencing (HepB core protein + insidecoil in BBa_J04450)
'''GFP modification'''
'''GFP modification'''
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*  colony PCR of the transformations from 6. September (PCR step 1 in pJET) - using pJET sequencing primers
*  colony PCR of the transformations from 6. September (PCR step 1 in pJET) - using pJET sequencing primers
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-> no band on the gel  
-> no band on the gel  
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* step 1 of the PCR reaction  
* step 1 of the PCR reaction  
 +
- With different template concentrations (from 20ng until 0.2ng added to the mix)  
- With different template concentrations (from 20ng until 0.2ng added to the mix)  
- With two different primer sets (both with the forward primer adding part of the coil + once with the GFP reverse primer adding a histag at the C-terminal and once with the sequencing reverse primer)
- With two different primer sets (both with the forward primer adding part of the coil + once with the GFP reverse primer adding a histag at the C-terminal and once with the sequencing reverse primer)
 +
-> bands of the expected size visible (even the samples that contain a very low ammount of template). There is also a second band visible (decreasing in strenght with decreasing template concentration)  
-> bands of the expected size visible (even the samples that contain a very low ammount of template). There is also a second band visible (decreasing in strenght with decreasing template concentration)  
-> something went wrong with the second set of reactions (the reactions where made in duplo)  
-> something went wrong with the second set of reactions (the reactions where made in duplo)  
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* Transformation With DH5α using different amount of ligation mixture for the electro transformation (1, 3 and 5 µL)
* Transformation With DH5α using different amount of ligation mixture for the electro transformation (1, 3 and 5 µL)
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-> more colonies where growing on the plates containing transformants transformed with 5µL
-> more colonies where growing on the plates containing transformants transformed with 5µL
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[[File:Colony_PCR12Sept.png]]
[[File:Colony_PCR12Sept.png]]
 +
 +
''Colony PCR 12 September''

Revision as of 15:28, 19 September 2012

week 20: 10 September - 16 September

Hepatitis B general

10 September

  • 2nd try colony PCR of the transformations from 4. September (with sequencing primers)

-> 6 out of 9 samples show an insert of the right size (see picture Colony PCR 12 September)


12 September

  • Miniprep and digestion check of colonies 5.Sept. (Hephis tag in BBa_J04450)

-> Restriction check shows inserts that might be too large (expected size: around 600bp) (see picture restriction check 12 September)

  • Sent samples for sequencing (HepB core protein + his tag in BBa_J04450)


Hepatitis B outside modification


  • 10 September (Kees)

• Colony PCR

The colonies were picked after which part of it was put in pcr-tubes for checking the content and part pas plated for continuation of the colony.

All the bands have a similar size of about 1100bp. The expected construct should contain a fragment of 1250bp. Since this is not the case and all the inserts are of the same size, the pcr failed.'



  • 11 September (Kees)

• Reverse PCR coil modification Same was done as on 7 September, with the following modifications: - 25 pcr cycles instead of 15. This will yield more template, taking the risk of modifications. - Analyse pcr result on a agarose gel before further processing. - Ligate overnight at 16 degrees if gel shows linear band at about 3kb. (protocols: http://www.neb.com/nebecomm/products_intl/faqproductM0202.asp, http://www.idtdna.com/pages/docs/user-guides-and-protocols/mutagenesis-application-guide.pdf, p.32) - Digest with DpnI after ligation


  • 12 September (Kees)

• Digestion and plating After digestion of the ligated sample and the non-ligated sample, the cells were transformed: - Template (positive control) - Ligated + digested - Digested only

  • 13 September (Kees)

One colony grew on the DpnI+Ligated plate. This colony will be checked with colony pcr and inocculated to produce anything that is in there.


Hepatitis B inside modification

10 September

  • 2nd try colony PCR of the transformations from 4. September (with sequencing primers)

-> one colony seems to have the correct insert in the pSB1C3 backbone -> no conclusion can be made about samples containing the Hep-inside coil fragment in the Bba_J04500 brick (see picture Colony PCR 12 September)


12 September

  • Miniprep and digestion check of colonies 5.Sept. (Hepinsidecoil in BBa_J04450)

-> Restriction check shows inserts that might be too large (expected size: around 600bp)

Restriction check 12.Sept.png

restriction check 12 September


  • Sent samples for sequencing (HepB core protein + insidecoil in BBa_J04450)

GFP modification

10 September

  • colony PCR of the transformations from 6. September (PCR step 1 in pJET) - using pJET sequencing primers

-> no band on the gel


11 September

  • step 1 of the PCR reaction

- With different template concentrations (from 20ng until 0.2ng added to the mix) - With two different primer sets (both with the forward primer adding part of the coil + once with the GFP reverse primer adding a histag at the C-terminal and once with the sequencing reverse primer)

-> bands of the expected size visible (even the samples that contain a very low ammount of template). There is also a second band visible (decreasing in strenght with decreasing template concentration) -> something went wrong with the second set of reactions (the reactions where made in duplo)


1st PCR step GFP-coil 11Sep.png


-> this shows that the experiment can be continued with the sample containing only 0.2ng template DNA (so the risk of transforming with the original template is lowered)

  • Of the GFP-coil 1st step (both primer sets) in pJET (samples with 0.2ng template in the PCR mixture)
  • Transformation With DH5α using different amount of ligation mixture for the electro transformation (1, 3 and 5 µL)

-> more colonies where growing on the plates containing transformants transformed with 5µL


12 September

  • Colony PCR of the transformations (GFP-coil step1 in pJET in DH5α)


Colony PCR12Sept.png

Colony PCR 12 September


-> the transformation was succesfull - there are colonies present that contain an insert with the expected size

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