Team:Heidelberg/Notebook modeling

From 2009.igem.org

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__NOTOC__
__NOTOC__
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=='''Multi-color Output'''==
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=='''Contents'''==
 +
 +
{| class="wikitable centered" border="2" rules="rows" width="600px" style="border-color:white;"
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|- 
 +
! Week                      !! colspan="7" |Days
 +
|-
 +
|style="text-align:center"| 32
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-3-2009|8-3-2009]]
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-4-2009|8-4-2009]]
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-5-2009|8-5-2009]]
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-6-2009|8-6-2009]]
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-7-2009|8-7-2009]]
 +
|style="text-align:center"| -
 +
|style="text-align:center"| -
 +
|-
 +
|style="text-align:center"| 33
 +
|style="text-align:center"| -
 +
|style="text-align:center"| -
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-12-2009|8-12-2009]]
 +
|style="text-align:center"| -
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-14-2009|8-14-2009]]
 +
|style="text-align:center"| -
 +
|style="text-align:center"| -
 +
|-
 +
|style="text-align:center"| 34
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-17-2009|8-17-2009]]
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-18-2009|8-18-2009]]
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-19-2009|8-19-2009]]
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-20-2009|8-20-2009]]
 +
|style="text-align:center"| [[Team:Heidelberg/Notebook_color_output#8-21-2009|8-21-2009]]
 +
|style="text-align:center"| -
 +
|style="text-align:center"| -
 +
|}
 +
 +
== 8-3-2009 ==
 +
 +
* got YFP/CFP-plasmids of Robert (Cellmembrane, ER, Cytosole)
 +
* Joel pledged to give us further color contructs (Cellmembrane, Mitochondria, Nucleus)
 +
 +
== 8-4-2009 ==
 +
 +
* transformation of all fluorescent proteins from upstairs (P.32-P.44 --> culture and miniprep tbd tomorrow)
 +
 +
 +
== 8-5-2009 ==
 +
 +
* transformation of all plasmids with targeted fluorescent proteins (P.32-P.43, P.44 still missing)
 +
* made bacterial stocks of p32-43 and placed in -80°C freezer
 +
 +
== 8-6-2009 ==
 +
 +
*Miniprep of p32, p33, p34, p35, p36, p37, p38, p39, p40, p41, p42, p43
 +
 +
== 8-7-2009 ==
 +
 +
* Ordered primers for: GPI-attachement signal, Ras1b, YFP, CFP, GFP, mi
 +
 +
== 8-12-2009 ==
 +
 +
* ordered primers for the mutagenesis PCR of the fluorescent proteins
 +
 +
 +
{| class="wikitable centered" border="2" rules="rows" width="450px" style="border-color:white;"
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|- style="background-color:#009ACD;"
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|style="text-align:center"|Plasmid ||style="text-align:center"| restriction site ||style="text-align:center"| mutagenesis position
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|- style="background-color:#63B8FF;"
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|style="text-align:center"| p41 ||style="text-align:center"| PstI || style="text-align:center"| 1257
 +
|- style="background-color:#63B8FF;"
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|style="text-align:center"| p43 || style="text-align:center"|PstI ||style="text-align:center"| 1001
 +
|- style="background-color:#63B8FF;"
 +
|style="text-align:center"| p37-Ras ||style="text-align:center"| PstI ||style="text-align:center"| 840
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|- style="background-color:#63B8FF;"
 +
|style="text-align:center"| p37 ||style="text-align:center"| NheI/EcoRI/PstI || style="text-align:center"|18/789/840
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|- style="background-color:#63B8FF;"
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|style="text-align:center"| p33 || style="text-align:center"|NheI/PstI/PstI/EcoRI || style="text-align:center"|34/259/799/805
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|- style="background-color:#63B8FF;"
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|style="text-align:center"| p35 || style="text-align:center"|NheI/NheI/PstI || style="text-align:center"|28/67/148
 +
|}
 +
 +
== 8-14-2009 ==
 +
 +
* two mutagenesis PCR's were done (p33g259a and p37a789g)
 +
 +
== 8-17-2009 ==
 +
 +
* the other mutagenesis PCR's were done
 +
* and all mutagenesis PCR products were transformed into DH5alpha bacterial cells
 +
 +
== 8-18-2009 ==
 +
 +
* no colony because of problems with the kanamycin plates
 +
* all mutagenesis PCR's were repeated
 +
*started to test the ligation orientation of inserts into BBb plasmids:
 +
- Digested p31 and a Jet insert with NheI and SpeI.
 +
-Ligated both together for 3 hrs at RT. (insert:vector= 1:1, 3:1, 1:3)
 +
-Transformed bacteria with ligate and plated them on Amp plates.
 +
-Repeated mutagenesis PCR again for (p33, p35, p37, p37-Ras, p41, p43)
 +
 +
== 8-19-2009 ==
 +
 +
* Ligation worked for all 3 plates. Picked 6 colonies for minipreping from each plate for analysis.
 +
* PCR amplification of each of GPI-attachement signal, EBFP alone, NLS alone and EBFP together with NLS.
 +
* Ran EBFP/NLS amplicons on gel and the run seemed to work.
 +
 +
<!--[[Image:p38_PCR_amplification_19-08-09.jpg|500px]]<!-- -->
 +
 +
'''''eBFP/NLS PCR amplification'''''
 +
* GPI-attachement signal was amplified overnight.
 +
 +
== 8-20-2009 ==
 +
* Restriction digestion of ligation products after minipreping.
 +
* Ran PCR products for GPI on gel--> nothing!:(
 +
* Restriction digestion showed only efficiency of 5% :( (only 1/18 digests showed insertion in the opposing direction)
 +
 +
== 8-21-2009 ==
 +
 +
* new mutagenesis PCR (without kit)
 +
 +
== 8-22-2009 ==
 +
 +
*Insert Amplification of mitoneet-eGFP by PCR
 +
 +
== 8-24-2009 ==
 +
 +
*Restriction digest of mutagenized Plasmids (PstI) and analysis on gel
 +
 +
*Amplified inserts were gel-purificated
 +
 +
<!-- [[Image:eBFP_NLS_und_eGFP_mitomeet.png|500px]]<!-- -->
 +
 +
 +
*What worked: eBFP, eBFP+NLS, eBFP_kozak, eBFP+NLS_kozak, eGFP, eGFP_kozak
 +
*What didn't: NLS, NLS_kozak, eGFP+mitomeet, mitomeet, eGFP+mitomeet_kozak, mitomeet_kozak,
 +
 +
== 8-26-2009 ==
 +
'''BBBing of Insertsequences'''
 +
 +
*PCR of cherry, cherry_myrpalm, myrpalm, NLS with kozak Primers to amplify cherry_kozak, cherry_myrpalm_kozak, myrpalm_kozak, NLS_kozak
 +
<!-- [[Image:08_26_09_kozak_von_cherry.jpg|500px]]<!-- -->
 +
 +
*Restriction with NheI and SpeI of localisationsequences and Flourophores, Restricted Plasmid was provided by Synthetic Promoter Group and digested with SAP
 +
*Ligation with p31
 +
*Transformation in DH5alpha with ligated Plasmids
 +
*Outplating of Transformed cells on Amp-plates
 +
 +
== 8-27-2009 ==
 +
 +
*Ligation and Transformation did not work (no colonies, except of two on the NLS )
 +
*New PCR with flourophores and localisationsequences, to get higher amounts
 +
<!--[[Image:08_27_09_insertamplification.png|500px]]<!-- -->
 +
 +
*GEl purification of: eGFP, eGFP_kozak, eBFP, eBFP_NLS, eBFP_kozak, eBFP_NLS_kozak, NLS_kozak, cherry, cherry_myrpalm, myrpalm, cherry_kozak, cherry_myrpalm_kozak, myrpalm_kozak
 +
 +
== 8-28-2009 ==
 +
'''BBBing of Insertsequences2.0'''
 +
 +
*Restrictiondigest of flourophores and localisationsequences with SpeI and NheI (1 h, Buffer 2, BSA)
 +
*Restrictiondigest of p49 with SpeI and NheI (1 h, Buffer 2, BSA) and SAP (30 min), purification
 +
*Nanodrop of digest shows no DNA inside of the samples -- purification was maybe unsuccessful
 +
 +
== 8-29-2009 ==
 +
 +
'''BBBing of Insertsequences2.1'''
 +
 +
*Restrictiondigest of flourophores and localisationsequences with SpeI and NheI (1 h, Buffer 2, BSA)
 +
*Restrictiondigest of p49 with SpeI and NheI (1 h, Buffer 2, BSA) and SAP (30 min), purification
 +
 +
== 8-31-2009 ==
 +
'''BBBing of Insertsequences2.1 (part 2)'''
 +
 +
*Ligation of Insertsequences with restricted p49
 +
*Transformation
 +
*Outplating -> Wrong resistance
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Revision as of 22:16, 18 October 2009



Multi-color Output

Contents

Week Days
32 8-3-2009 8-4-2009 8-5-2009 8-6-2009 8-7-2009 - -
33 - - 8-12-2009 - 8-14-2009 - -
34 8-17-2009 8-18-2009 8-19-2009 8-20-2009 8-21-2009 - -

8-3-2009

  • got YFP/CFP-plasmids of Robert (Cellmembrane, ER, Cytosole)
  • Joel pledged to give us further color contructs (Cellmembrane, Mitochondria, Nucleus)

8-4-2009

  • transformation of all fluorescent proteins from upstairs (P.32-P.44 --> culture and miniprep tbd tomorrow)


8-5-2009

  • transformation of all plasmids with targeted fluorescent proteins (P.32-P.43, P.44 still missing)
  • made bacterial stocks of p32-43 and placed in -80°C freezer

8-6-2009

  • Miniprep of p32, p33, p34, p35, p36, p37, p38, p39, p40, p41, p42, p43

8-7-2009

  • Ordered primers for: GPI-attachement signal, Ras1b, YFP, CFP, GFP, mi

8-12-2009

  • ordered primers for the mutagenesis PCR of the fluorescent proteins


Plasmid restriction site mutagenesis position
p41 PstI 1257
p43 PstI 1001
p37-Ras PstI 840
p37 NheI/EcoRI/PstI 18/789/840
p33 NheI/PstI/PstI/EcoRI 34/259/799/805
p35 NheI/NheI/PstI 28/67/148

8-14-2009

  • two mutagenesis PCR's were done (p33g259a and p37a789g)

8-17-2009

  • the other mutagenesis PCR's were done
  • and all mutagenesis PCR products were transformed into DH5alpha bacterial cells

8-18-2009

  • no colony because of problems with the kanamycin plates
  • all mutagenesis PCR's were repeated
  • started to test the ligation orientation of inserts into BBb plasmids:

- Digested p31 and a Jet insert with NheI and SpeI. -Ligated both together for 3 hrs at RT. (insert:vector= 1:1, 3:1, 1:3) -Transformed bacteria with ligate and plated them on Amp plates. -Repeated mutagenesis PCR again for (p33, p35, p37, p37-Ras, p41, p43)

8-19-2009

  • Ligation worked for all 3 plates. Picked 6 colonies for minipreping from each plate for analysis.
  • PCR amplification of each of GPI-attachement signal, EBFP alone, NLS alone and EBFP together with NLS.
  • Ran EBFP/NLS amplicons on gel and the run seemed to work.


eBFP/NLS PCR amplification

  • GPI-attachement signal was amplified overnight.

8-20-2009

  • Restriction digestion of ligation products after minipreping.
  • Ran PCR products for GPI on gel--> nothing!:(
  • Restriction digestion showed only efficiency of 5% :( (only 1/18 digests showed insertion in the opposing direction)

8-21-2009

  • new mutagenesis PCR (without kit)

8-22-2009

  • Insert Amplification of mitoneet-eGFP by PCR

8-24-2009

  • Restriction digest of mutagenized Plasmids (PstI) and analysis on gel
  • Amplified inserts were gel-purificated


  • What worked: eBFP, eBFP+NLS, eBFP_kozak, eBFP+NLS_kozak, eGFP, eGFP_kozak
  • What didn't: NLS, NLS_kozak, eGFP+mitomeet, mitomeet, eGFP+mitomeet_kozak, mitomeet_kozak,

8-26-2009

BBBing of Insertsequences

  • PCR of cherry, cherry_myrpalm, myrpalm, NLS with kozak Primers to amplify cherry_kozak, cherry_myrpalm_kozak, myrpalm_kozak, NLS_kozak
  • Restriction with NheI and SpeI of localisationsequences and Flourophores, Restricted Plasmid was provided by Synthetic Promoter Group and digested with SAP
  • Ligation with p31
  • Transformation in DH5alpha with ligated Plasmids
  • Outplating of Transformed cells on Amp-plates

8-27-2009

  • Ligation and Transformation did not work (no colonies, except of two on the NLS )
  • New PCR with flourophores and localisationsequences, to get higher amounts
  • GEl purification of: eGFP, eGFP_kozak, eBFP, eBFP_NLS, eBFP_kozak, eBFP_NLS_kozak, NLS_kozak, cherry, cherry_myrpalm, myrpalm, cherry_kozak, cherry_myrpalm_kozak, myrpalm_kozak

8-28-2009

BBBing of Insertsequences2.0

  • Restrictiondigest of flourophores and localisationsequences with SpeI and NheI (1 h, Buffer 2, BSA)
  • Restrictiondigest of p49 with SpeI and NheI (1 h, Buffer 2, BSA) and SAP (30 min), purification
  • Nanodrop of digest shows no DNA inside of the samples -- purification was maybe unsuccessful

8-29-2009

BBBing of Insertsequences2.1

  • Restrictiondigest of flourophores and localisationsequences with SpeI and NheI (1 h, Buffer 2, BSA)
  • Restrictiondigest of p49 with SpeI and NheI (1 h, Buffer 2, BSA) and SAP (30 min), purification

8-31-2009

BBBing of Insertsequences2.1 (part 2)

  • Ligation of Insertsequences with restricted p49
  • Transformation
  • Outplating -> Wrong resistance
Retrieved from "http://2009.igem.org/Team:Heidelberg/Notebook_modeling"