Team:Freiburg Bioware/NoteBook/Labjournal/October

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<br/><br/><br/><br/><br/><br/><br/>
<br/><br/><br/><br/><br/><br/><br/>
<b>Conclusion:</b> The VP2/3 fusion particle was transcribed and translated AND was transported back into the nucleus in order to be packaged by the ITR-flanked gene of interest. <br/>
<b>Conclusion:</b> The VP2/3 fusion particle was transcribed and translated AND was transported back into the nucleus in order to be packaged by the ITR-flanked gene of interest. <br/>
 +
 +
====<p style="font-size:15px; background-color:#66bbff;"><b>Cloning of VP2/3_BAP_HSPG-KO and VP2/3_His_HSPG-KO into pCerulean_Zegfr:1907_Middlelinker and VP2/3_His_HSPG-KO into pCerulean_VP1up_NLS_mVenus</b></p>====
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<b>Investigator: Stefan </b><br>
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<p style="font-size:13px; color:red;"></p>
 +
 +
Vector name:
 +
<ul>
 +
<li>pCerulean_Zegfr:1907_Middlelinker (P408)</li>
 +
<li>pCerulean_VP1up_NLS_mVenus (P426)</li>
 +
</ul>
 +
Insert name:
 +
<ul>
 +
<li>pBS1C3_001_VP2/3_587_BAP_HSPG-KO clone1 (P660)</li>
 +
<li>pBS1C3_001_VP2/3_587_BAP_HSPG-KO clone2 (P661)</li>
 +
<li>pBS1C3_001_VP2/3_587_His_HSPG-KO clone1 (P662)</li>
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<li>pBS1C3_001_VP2/3_587_His_HSPG-KO clone2 (P663)</li>
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</ul>
 +
 +
<br />
 +
{| border="1"
 +
| '''components'''  || align="right" |'''volume for inserts (P660 - P663)  /µl'''|| align="right" |'''volume of P408  /µl''' || align="right" |'''volume of P426 /µl'''
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|-
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| DNA  ||  align="right" |10 ||  align="right" |3||  align="right" |3
 +
|-
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| BSA (10x) ||  align="right" |3 ||  align="right" |2||  align="right" | 2
 +
|-
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| Buffer 4 (10x)||  align="right" |3 ||  align="right" |2 ||  align="right" | 2
 +
|-
 +
|Enzyme NgoMIV||  align="right" |1||  align="right" |- ||  align="right" |-
 +
|-
 +
|Enzyme PstI||  align="right" |1||  align="right" |1 ||  align="right" |1
 +
|-
 +
|Enzyme MscI||  align="right" |1||  align="right" |- ||  align="right" |-
 +
|-
 +
|Enzyme AgeI||  align="right" |-||  align="right" |1 ||  align="right" |1
 +
|-
 +
|H2O||  align="right" |11||  align="right" |11||  align="right" |11
 +
|-
 +
|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | 30||  align="right" | 20||  align="right" |20
 +
|}
 +
 +
<br />
 +
<b>Gel:</b><br />
 +
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt<br />
 +
 +
 +
<br/>
 +
[[Image:Freiburg10 digestion cloning BAP His to Affi Middle.jpg|450px|]]<br/>
 +
 +
<br/>
 +
 +
 +
<b>Gel extraction</b>: <br>
 +
Was performed according to protocol.
 +
 +
<br>
 +
<b>T4 Ligation</b>: <br>
 +
{| border="1"
 +
|ligation name || align="right" |408 + 660|| align="right" |408 + 661|| align="right" |408 + 662|| align="right" |408 + 663|| align="right" |426 + 662|| align="right" |426 + 663
 +
|-
 +
|volume of vector || align="right" |3,4 || align="right" | 3,05|| align="right" | 3,35|| align="right" | 2,56|| align="right" | 2,74|| align="right" | 3,13
 +
|-
 +
|volume of insert|| align="right" |4,6 || align="right" |4,95|| align="right" |4,65|| align="right" |5,44|| align="right" |5,26|| align="right" |4,87
 +
|-
 +
|}
 +
 +
<br>
 +
<b>Transformation</b>: <br>
 +
Was performed according to standard protocol using BL21 cells.
 +
<br/>
 +
<html>
<html>
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Revision as of 21:45, 1 October 2010

Contents

136. labday 01.10.2010

mini preps of CD clones

Investigator: Kira

c(p689)= 115 ng/ul
c(690)= 151, 20 ng/ul
c(691)= 122,27 ng/ul
c(p692) = 126,42 ng/ul

test digestion of CD clones

Investigator: Kira

Components sample Volume/µL
DNA 4,0 µl
BSA (10x) 2 µl
Buffer no. 4 2,0 µl
Enzyme 1 XbaI 1,0 µl
Enzyme 2 AgeI 1,5 µl
H2O 9,5 µl
Total volume 20


incubation @ 37 C for approx. 2 h

1% agarose gel

Freiburg10 CD testdigestion 2010-10-01.jpg

Biobrick assembly pSB1C3_lITR_hTERT_beta-globin_CD

Investigator: Kira

c(pSB1C3_lITR_hTERT_beta-globin)= 333 ng/ul
c(pSB1C3_CD)= 151 ng/ul

Components vector Volume/µL insert Volume/µL
DNA 4,5 µl 6
BSA (10x) 3 µl 3
Buffer no. 4 3,0 µl 3
Enzyme 1 XbaI 0 µl1,5
Enzyme 2 SpeI 1,5 µl 0
Enzyme 3 PstI-HF 1,0 1
H2O 17 15,5
Total volume 25


incubation @ 37 C for approx. 2 h

1% agarose gel

Freiburg10 CD-beta-globin.jpg

Ligation

DNA-mix: 8 ul (vector 4,6ul)+(insert 3,4 ul)
T4 ligase: 1 ul
T4 buffer: 1 ul
Incubation @ RT for 30 min

Transformation was performed according to the standard protocol w BL21 cells.


Sequencing results of pCerulean_Zegfr:1907_MiddleLinker_VP2/3_HSPG-KO

Investigator: Hanna

Comment: All N-terminal fusion approaches with VP2/3_HSPG-KO revealed positive results except of pCerulean_Zegfr:1907_MiddleLinker_VP2/3_HSPG-KO. Another clone was picked, preped, test digested and sent for sequencing.

Freiburg10 pCerulean Zegfr1907 ML VP23HSPGKO.png


Conclusion: Sequencing results revealed positive results.

Picking clones of pGA14_MiddleLinker_VP2/3_insCap and pGA14_MiddleLinker_VP2/3_HSPG-KO

Investigator: Hanna

Unfortunately there grew a bacteria lawn over night - it was hardly not possible to pick clones. Nevertheless I tried and picked 2 clones of pGA14_MiddleLinker_VP2/3_insCap and pGA14_MiddleLinker_VP2/3_HSPG-KO.
To do: Mini-Prep and test digestion.

Sequencing results of pSB1C3_001_VP2/3_587-KO_BAP and pSB1C3_001_VP2/3_587-KO_6xHis

Investigator: Hanna

Comment: For the creation of our super constructs, the His-Tag and the BAP motif need to be cloned into VP2/3 for N-terminal fusion to VP2. Sequencing results showed that the 6xHis and BAP motif was not cloned into VP2/3_insCap.

Sample gallery
Freiburg10 Seq BAP his.png
Freiburg10 Seq BAP his2.png


To do: Clone 587-KO_6xHis and 587-KO_BAP into pSB1C3_001_VP2/3_insCap 1. via digestion of inserts and 2. via hybridization of referring oligos - digestion of vector with 800-900 ng.

Impressions of transfection of AAV293 with pCerulean_VP1up_NLS_mVenus_VP2/3

Investigator: Adrian

Yesterday AAV293 cells were transfected with pCerulean_VP1up_NLS_mVenus_VP2/3 and GMK-TK30 as gene of interest. Today, nuclear localization of the produced mVenus_VP2/3 fusion protein was visible and can be nicely seen in the following pictures:

Freiburg10 VP mVenus fusion localisation 5.jpg
Freiburg10 VP mVenus fusion localisation 7.jpg

















Conclusion: The VP2/3 fusion particle was transcribed and translated AND was transported back into the nucleus in order to be packaged by the ITR-flanked gene of interest.

Cloning of VP2/3_BAP_HSPG-KO and VP2/3_His_HSPG-KO into pCerulean_Zegfr:1907_Middlelinker and VP2/3_His_HSPG-KO into pCerulean_VP1up_NLS_mVenus

Investigator: Stefan

Vector name:

  • pCerulean_Zegfr:1907_Middlelinker (P408)
  • pCerulean_VP1up_NLS_mVenus (P426)

Insert name:

  • pBS1C3_001_VP2/3_587_BAP_HSPG-KO clone1 (P660)
  • pBS1C3_001_VP2/3_587_BAP_HSPG-KO clone2 (P661)
  • pBS1C3_001_VP2/3_587_His_HSPG-KO clone1 (P662)
  • pBS1C3_001_VP2/3_587_His_HSPG-KO clone2 (P663)


components volume for inserts (P660 - P663) /µlvolume of P408 /µl volume of P426 /µl
DNA 10 33
BSA (10x) 3 2 2
Buffer 4 (10x)3 2 2
Enzyme NgoMIV1- -
Enzyme PstI11 1
Enzyme MscI1- -
Enzyme AgeI-1 1
H2O111111
Total volume (e.g. 15,20,25,30 µl) 30 2020


Gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt



Freiburg10 digestion cloning BAP His to Affi Middle.jpg



Gel extraction:
Was performed according to protocol.


T4 Ligation:

ligation name 408 + 660408 + 661408 + 662408 + 663426 + 662426 + 663
volume of vector 3,4 3,05 3,35 2,56 2,74 3,13
volume of insert4,6 4,954,655,445,264,87


Transformation:
Was performed according to standard protocol using BL21 cells.

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