Team:Freiburg Bioware/NoteBook/Labjournal/September

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Contents

September

107. labday 01.09.2010

Loop insertion BioBricks: Sequencing results

Investigator: Achim, Anna

Comment: Sequencing results of Loop insertion BioBricks from 31.08.10; the following clones were sent for sequencing:


  • 2: 453 BAP: No Insert

Freiburg10 453 bap.JPG

  • 3:453 His: Insert okay

Freiburg10 453 his.JPG

  • 5: 453 RGD: Mutation in 453 upstream region

Freiburg10 453 rgd.JPG

  • 7: 587 BAP: Mutation in Bap insert

Freiburg10 587 bap.JPG

  • 9:587 His: Insert okay

Freiburg10 587 his.JPG

  • 11:587 KO BAP : Insert okay

Freiburg10 587 KO bap.JPG

  • 13:587 KO empty : Insert okay

Freiburg10 587 KO empty.JPG

  • 16: 587 KO his: Mutation in 587 KO

Freiburg10 587 ko his.JPG

  • 17: 587 KO rgd: No insert

Freiburg10 587 KO rgd.JPG

  • 19 :587 RGD: Insert okay

Freiburg10 587 rgd.JPG

  • 25: 587 Z34C: Mutation in downstream 587 region

Freiburg10 587 z34c.JPG

Mini-Preps:

New Mini-Preps of the samples with no/mutated insert were prepared. The following clones were used:

Components 453 BAP 587 BAP 453 RGD 587 KO RGD 587 KO HIS 453 Z34C 587 Z34C 587 KO Z34C 587 KO Z34C SPACER
Clone1.3 + 1.4 2.3 + 2.4 4.3 + 4.4 6.3 + 6.4 9.3 + 9.4 11.3 + 11.412.2 + 12.4 13.2 + 13.4 14.3 + 14.4


Comment: Samples 1.3, 2.3, 4.3, 6.3, 9.3, 11.3, 12.2, 13.2, 14.3 were sent for sequencing. For sequencing results go to labday 02.09.10.

.

Design of primers for pAAV_RC

Investigator: Bea

Primers for different cloning strategies of the synthesized cap gene into pAAV_Rc have been designed.

  1. The first primers are mutagenesis primers which delete the KpnI recognition site at position 3968. These primers can (or should be used) be used if cloning with BspMI/BfuI (isochizomer) does not cut the pAAV_RC efficiently. Instead of using this enzyme we can use XcmI to subclone the cap gene with performing a site-directed mutagenesis afterwards with designed primers.
  2. The second idea is to design oligoduplexes (hybridized oligos) which contain the recognition site for BspMI. In Gormley et al (2002) the idea was to add oligodupexes with the recognition site for BspMI and therefore provide the "second recognition site" in trans.

Next step: Check primers and order them today! Designed primers are stored in my Workingfolder under paav_rc and a word document was created whcih can be found under Oligos --> primers for different strategies...

Sequencing results of pSB1C3_Zegfr:1907_Linker

Investigator: Hanna

Comment: Sequencing results looked all well. One bp was missing in the theoretical sequence of SEG suffix - but is correct in the actual sequence.


pSB1Cr_Zegfr:1907_LongLinker:
PSB1C3 Zegfr1907 LongLinker.JPG

pSB1Cr_Zegfr:1907_SEG:
PSB1C3 Zegfr1907 SEG.JPG

pSB1Cr_Zegfr:1907_MiddleLinker:
PSB1C3 Zegfr1907 MiddleLinker.JPG

pSB1Cr_Zegfr:1907_ShortLinker:
PSB1C3 Zegfr1907 ShortLinker.JPG

These constructs will be cloned into pCerulean for N-terminal fusion to VP2.
go to the top ;)

Trafo evalutaion and Inoculation

Investigator: Kira
The plate with transformed CD-biobrick contains around 40 colonies. 4 of them will be inoculated into LB and incubated @37 C over-night.
go to the top ;)

Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)

Investigator: Patrick


The two Ligations were performed according to the standard protocol:
1 µl T4 DNA ligase, 1 µl 10x Buffer, 4,6 µl vector (P273), 3,34 µl Insert (P276, upper and lower band).
Incubation time: 50 minutes.

During the transformation i forgot to put the cells into the 37°C shaker so i incubated them afterwards again for 45 minutes at 37 °C on a shaker.

The mutual conles will be picked tomorrow to inoculate 10 ml DYT following a mini-prep on 03.09.2010

Transfection, Seeding AAV293 and HT1080

Investigator: Kerstin

  • Transfection: 10 plates, same treatment (10µg of RC (P357), pHelper (P356)and mVenus (P263), following the standard protocol)
  • Seeding HT1080 for transduction with virus from 21.08.2010 (10µg of RC, pHelper, TKGMK; pH=7,10)
  • Seeding AAV293 for transfection with plasmids without HgH or beta-globin (verifying functionality)

Cloning of ZEGFR:1907_Middle-Linker,ZEGFR:1907_Short-Linker, ZEGFR:1907_SEG-Linker and ZEGFR:1907_Long-Linker into pCerulean

Investigator: Stefan

Comment:


Digestion:

components Vector (P273) ZEGFR:1907_Middle-Linker (P290) ZEGFR:1907_Short-Linker (P292) ZEGFR:1907_SEG-Linker (P296) ZEGFR:1907_Long-Linker (P298)
DNA 3,8 8,8 10,2 9,2 8,7
BSA (10x) 2 2 2 2 2
Buffer 4 (10x)2 2 2 2 2
XbaI 1 1111
PstI 11111
H2O10,2 5,2 3,8 4,8 5,3
Total volume 20 20 20 20 20


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



Freiburg10 pCerulean affi linker.jpg




Gelextraction:

The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

  • P273: c= 3,1 ng/µl
  • P290: c= 3,2 ng/µl
  • P292: c= 1,8 ng/µl
  • P296: c= 3,0 ng/µl
  • P298: c= 3,2 ng/µl


T4 Ligation:

The Ligation was performed as following:

Comment: No sufficent vector concentration, therefore 5 µl of vector was used for every approach.

  • Vector Volume: 5 µl
  • Insert Volume: 3 µl


  • 1µl T4-Ligase buffer (10x)
  • 8µl (Vector + Insert) mix
  • 1µl T4-Ligase


Incubating for 40 minutes.


Transformation:

Trafo was performed according to the standard protocol (BL21). The cells were plated on a agar plate with chloramphenicol


108. labday 02.09.2010

Sequencing of loop insertions BioBricks: 2nd round

Investigator: Achim, Anna

We analyzed the new sequencing results and found two correct sequences:

  • 587 BAP clone 2.3
  • 453 RGD clone 4.3

The other samples didn't contain any inserts or had wrong insertions. Apparently, the vector tends to religate easily. A possible reason for the religation of the incompatible ends is the overnight ligation at 18°C. We sent preps of the remaining 7 ligations for sequencing and will prep new clones or repeat the ligation tomorrow, depending on the new sequences.

Comment: Clones 1.4, 6.4, 9.4, 11.4, 12.4, 13.4, 14.4 were sent for sequencing. For sequencing results go to labday 03.09.10.

.

Mini-preps of pSB1C3_SV40 and pCerulean_VP1_NLS

Investigator: Bea (Chris L)

Mini-Prep was performed according to the standard protocol

  • P363 = pSB1C3_SV40 clone 1 = 277,70 ng/µl
  • P364 = pSB1C3_SV40 clone 2 = 265,01 ng/µl
  • P365 = pCerulean_VP1up_NLS (1) clone 1 = 473,14 ng/µl
  • P366 = pCerulean_VP1up_NLS (1) clone 2 = 455,88 ng/µl
  • P367 = pCerulean_VP1up_NLS (1) clone 3 = 482,86 ng/µl
  • P368 = pCerulean_VP1up_NLS (2) clone 1 = 438,52 ng/µl
  • P369 = pCerulean_VP1up_NLS (2) clone 2 = 478,00 ng/µl
  • P370 = pCerulean_VP1up_NLS (2) clone 3 = 419,00 ng/µl
    Construct were digested with XbaI and PstI for 45 minutes at 37°C. The loading plan on the 1% agarose gel looks like this:

    _M_ ___ _363_ ___ _364_ _365_ _366_ _367_ _368_ _369_ _370_ ___

    Results: P365 was sent for sequencing because test digestion looks goos. In contrast to the SV40 appraoch. This needs to be repeated tomorrow.
    • Plasmid: pCerulean_VP1up_NLS
    • Plasmid number: P365
    • Tube name: SB1
    • Primer used: CMV-F

    Mini-Prep and test digestion of pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker

    Investigator: Hanna

    Comment: Zegfr:1907 (Affibody) and the His-Tag which was coupled to the middle linker were cloned into pCerulean. 3 clones of each construct were picked yesterday.


    Plasmid Mini-Prep

    • new vector name: pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker


    Glycerol Stocks
    1. pCerulean_Zegfr:1907

    Clone 1 Clone 2 Clone 3
    Bacteria strain XL1b XL1b XL1b
    Plasmidname pCerulean_Zegfr:1907 pCerulean_Zegfr:1907 pCerulean_Zegfr:1907
    Date 2.9.10 2.9.10 2.9.10
    given glycerol-stock no. B275 B276 B277
    given plasmid no. P371 P372 P373


    2. pCerulean_6xHis_MiddleLinker

    Clone 1 Clone 2 Clone 3
    Bacteria strain XL1b XL1b XL1b
    Plasmidname pCerulean_6xHis_MiddleLinker pCerulean_6xHis_MiddleLinker pCerulean_6xHis_MiddleLinker
    Date 2.9.10 2.9.10 2.9.10
    given glycerol-stock no. B278 B279 B280
    given plasmid no. P374 P375 P376


    Test digestion

    • buffer used: 4; Restriction-enzymes used: Enzyme 1 PstI-HF ; Enzyme 2 EcoRI-HF
    • Plasmid
      • Given Plasmid-Number: 371; DNA concentration: 381.3 ng/µL;
      • Given Plasmid-Number: 372; DNA concentration: 377.5 ng/µL;
      • Given Plasmid-Number: 373; DNA concentration: 409.7 ng/µL;
      • Given Plasmid-Number: 374; DNA concentration: 404.2 ng/µL;
      • Given Plasmid-Number: 375; DNA concentration: 366.1 ng/µL;
      • Given Plasmid-Number: 376; DNA concentration: 375.0 ng/µL;


    Comments::)

    Pipetting scheme for each test digestion:

    Components Volume/µL
    DNA 2.7
    BSA (10x) 1
    Buffer no. 4 (10x) 1
    EcoRI-HF 0.5
    PstI-HF 0.5
    H2O 4.3
    Total volume 10


    • Incubation: 55 minutes


    Agarose-Gel:


    0.875 g Agarose, 50 mL TAE (1.75 %), 3 µL EthBr, at 115 Volt, running time: 35 minutes

    Sample Sample/µl] Loading dye (6x)/µl Expected size
    pCerulean_Zegfr:1907 clone 1 10 µl 2 µl 231 bp
    pCerulean_Zegfr:1907 clone 2 10 µl 2 µl 231 bp
    pCerulean_Zegfr:1907 clone 3 10 µl 2 µl 231 bp
    pCerulean_6xHis_MiddleLinker clone 1 10 µl 2 µl 105 bp
    pCerulean_6xHis_MiddleLinker clone 2 10 µl 2 µl 105 bp
    pCerulean_6xHis_MiddleLinker clone 3 10 µl 2 µl 105 bp


    • Marker: GeneRuler ladder mix
    Marker /µL Sample P371 /µl Sample P372 /µl Sample P373 /µl Sample P374 /µl Sample P375 /µl Sample P376 /µl
    Lane 5 12 12 12 12 12 12


    Comments: Test digestion looked well:

    Freiburg10 pCerulean Zegfr1907andpCerulean 6xHis ML.png
    Clone 1 of each construct were sent for sequencing (P371 and P374 = HW1 and HW2). Used primer: GATC_std_CMV-F.

    Midi-Prep of pSB1C3_lITR_CMV_mVenus_hGH_rITR clone1 & pSB1C3_lITR_CMV_beta-globin_mVenus_rITR clone1

    Investigators: Chris W.

    Midi-Preps of pSB1C3_lITR_CMV_mVenus_hGH_rITR clone1=P377 and pSB1C3_lITR_CMV_beta-globin_mVenus_rITR clone1=P378

    The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

    plasmid-no. P377P378
    concentration (ng/µl)325,04 561,15


    Mini-Prep and test digestion of the CD biobricks

    Investigator: Kira

    Mistake.png
    Ohne Worte *lach*

    Wh did you digest with eco and ndeI?? is there a spechía reasonn for it?? if the coonstruct was in the rfc standard could digest it with the igem standard enzymes??

    components sample /µl
    DNA 2,5
    BSA (100x) 0
    Buffer ___4_ (10x) 1,5
    Enzyme NdeI (no.Lab:___) 0,5
    Enzyme EcoRI (no.Lab:___)0,5
    H2O10
    Total volume (e.g. 15,20,25,30 µl) |15
  • Incubation: 1 h

    Freiburg10 test digestion of CD.jpg

    Sample 1 was sent for sequencing

    109. labday 03.09.2010

    Miniprep and test digestion of pSB1C3_lITR_pTERT_ßglobin_mVenus_hGH_rITR

    Investigator: Achim

    Comment: The test digestion did not show the expected fragments of 2000 and 2500 bp. the photo was exposed too long to see distinct bands. Digestion will be repeated.

    AA1.png

    Sequencing results

    Investigator: Hanna

    Comment: pCerulean_Zegfr:1907 and pCerulean_6xHis_MiddleLinker were cloned and sent for sequencing yesterday.

    1. pCerulean_Zegfr:1907: Sequencing looked well.
    Freiburg10 pCerulean Zegfr1907 seq.JPG

    2. pCerulean_6xHis_MiddleLinker: Sequencing looked also well. Freiburg10 pCeruelan 6xHis MiddleLinker seq.JPG

    Cloning of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

    Investigator: Hanna

    Comment: For N-terminal fusion to VP2, the Affibody - fused to different kinds of linkers - will be cloned into the expression plasmid pCerulean. For imaging CFP, which was fused to the middle linker, will be cloned into pCerulean.



    Practical Cloning:

    • plasmid:
      • Vector: name: pCerulean number: P273
      • Insert: name: pSB1C3_Zegfr:1907_LongLinker (P298), pSB1C3_Zegfr:1907_MiddleLinker (P290), pSB1C3_Zegfr:1907_SEG (P296), pSB1C3_Zegfr:1907_ShortLinker (P292), pSB1C3_CFP_MiddleLinker (P276)
    • new vector name: pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker
    • buffer used: 4 ; Restriction-enzymes used: Enzyme 1 EcoRI-HF ; Enzyme 2 PstI-HF
    • DNA concentration (vector): 419.5 ng/µL ; DNA concentration (insert): P298 229.7 ng/µL, P290 227.4 ng/µl; P296 218.7 ng/µL; P292 196.6 ng/µL; P276 207.7 ng/µL


    Comments: No.

    Digestion

    components volume of pCerulean /µl volume of Zegfr:1907_MiddleLinker /µl volume of CFP_MiddleLinker /µL
    DNA 8.3 7.6 4.8
    BSA (10x) - - -
    Buffer 4 (10x) 2 2 2
    Enzyme 1 EcoRI-HF 1 1 1
    Enzyme 2 PstI-HF 1 1 1
    H2O 7.7 8.4 11.2
    Total volume 20 20 20
    • Incubation: 1.5 h



    Agarose-Gel:


    0.8 g Agarose, 50 TAE (1.5 %), 5 µL EthBr, at 70 Volt, running time: ~ 1 hour

    Sample Sample/µl] Loading dye (6x)/µl Expected size 1
    P273 20 µl 4 µl 3922 bp
    P298 20 µl 4 µl 273 bp
    P290 20 µl 4 µl 261 bp
    P296 20 µl 4 µl 345 bp
    P292 20 µl 4 µl 249 bp
    P276 20 µl 4 µl 801 bp


    • Marker: GeneRuler ladder mix
    Marker Sample P273 /µl Sample P276 /µl Sample P290 /µl Sample P292 /µl Sample P296 /µl Sample P298 /µl
    Lane 5 24 24 24 24 24 24



    Gel extraction


    Gel measurement:

    Sample Volume Concentration
    P273 20 115.45 ng/µL
    P276 20 9.91 ng/µL
    P298 20 5.07 ng/µL
    P296 20 9.03 ng/µL
    P290 20 10.31 ng/µL
    P292 20 20.78 ng/µL




    Ligation


    1. PCerulean_Zegfr:1907_LongLinker

    P298 P273
    Volume/µl 6.61 1.39


    2. PCerulean_Zegfr:1907_MiddleLinker

    P290 P273
    Volume/µl 5.53 2.47


    3. PCerulean_Zegfr:1907_SEG

    P296 P273
    Volume/µl 6.17 1.83


    4. PCerulean_Zegfr:1907_ShortLinker

    P292 P273
    Volume/µl 4.11 3.89


    5. PCerulean_CFP_MiddleLinker

    P276 P273
    Volume/µl 7.02 0.98


    Trafo


    Trafo was performed following the standard protocol. Used cells: XL1b, DNA amount: 2.5 µL of each ligation reaction. Plates were stored @ 37°C room over night.

    Harvest viral particles, Seeding HT1080 cells

    Investigator: Adrian, Kerstin

    • Harvest viral particles of Transfection from 31.08.2010 (six approaches: 10µg of RC (2x P326 and 2x P325 and 2x Stratagene), pHelper and GOI (P262))
    • Seeding HT1080 cells

    Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273)

    Investigator: Patrick


    The Miniprep was performed according to the standard protocol. Labelling:

    • P381: pCerulean_middlelinker clone 1 upper band : 526,0 ng/µl
    • P382: pCerulean_middlelinker clone 2 upper band : 522,0 ng/µl
    • P383: pCerulean_middlelinker clone 3 upper band : 463,0 ng/µl
    • P384: pCerulean_middlelinker clone 1 lower band : 465,0 ng/µl
    • P385: pCerulean_middlelinker clone 2 lower band : 500,0 ng/µl
    • P386: pCerulean_middlelinker clone 3 lower band : 524,0 ng/µl
    Öhm Patrick: Hier ist der Grund, warum das Experiment "wiederholt" wurde: Falsche Beschrieftung hier, als auch in der Excel Tabelle ;) "Finde den Fehler!" :P


    see also http://www.molbiotech.uni-freiburg.de/iGEM/wiki2010/index.php/August_2010#Cloning_CFP_middlelinker_.28from_pSB1C3_CFP_middlelinker.2C_P276.29_into_pCerulean_.28P273.29

    ;-) bitte ausfüllen

    Testdigestion: 7 µl DNA sample, 1 µl Buffer 4 (10x), 1 µl BSA, 0,5 µl Xba, 0,5 µl PstI-HF
    Expected size of the fragments: 786 & 2053bp
    There seems to be something wrong.

    Freiburg10 pat20100903.jpg

    Sent for sequencing: P381 & P385 Labelling: P381-CMV_rev & P385-CMV_rev
    Used primer: O21 : CMV_reverse_qPCR

    Cloning of pSB1C3_leftITR_pTert and pSB1C3_mGMK with pSB1C3_leftITR_CMV

    Investigator: Chris L.

    • Vector: name: pSB1C3_leftITR_pTERT clone 1 P256 c=179,2 ng/µl
    • Vector: name: pSB1C3_leftITR_CMV P188 c=231,6 ng/µl
    • Insert: name: pSB1C3_mGMK P195 c=258,6 ng/µl
    • new vector name: pSB1C3_leftITR_CMV_GMK
    • new vector name: pSB1C3_leftITR_pTERT_GMK
    • buffer used: 4 ; Restriction-enzymes used: Enzyme 1 PstI ; Enzyme 2 SpeI ; Enzyme 3 XbaI


    components P195 P256 /µl P188 /µl
    DNA 7,7 11,2 8,6
    BSA (10x) 2 2 2
    Buffer 4 (10x)2 2 2
    Enzyme SpeI 1 1 0
    Enzyme XbaI 0 0 1
    Enzyme PstI 1 1 1
    H2O 6,3 2,8 5,4
    Total volume (e.g. 15,20,25,30 µl) 2020 20


    0,5 g Agarose,50 ml TAE (1%), 3 µl GELRED , at 130 Volt, running time:45

    Results:
    Freiburg10 pSB1C3 lITR pTERT and pSB1C3 lITR CMV with GMK Insert.jpg

    • c(Insert)= 15,37 ng/µl; size: 627 bp
    • c(Vector)= 50,84 ng/µl; size: 2869 bp
    • c(Vector)= 75,64 ng/µl; size: 2672 bp


    • Ligation of PCR products and vector:

    For the Ligation 1µl T4 buffer (10x) and 1µl T4 ligase were used. Incubation time: 45 min

    pSB1C3_leftITR_CMV + GMK /µl pSB1C3_leftITR_pTERT + GMK
    Vector 5,48 6,21
    Insert 2,52 1,79


    • Transformation:

    The transformation was done following the standard protocol using XL1 blue cells.

    Sequence analysis of pCerulean_VP1up

    Investigator: Bea

    The assembled pCerulean_VP1up which serves as the first construct in the VP1 insertion cloning assembly was sent for sequencing.

    • Plasmid used: P365
    • Primer used: CMV-F
    • Tube name: SB1
    • Folder (Geneious): N-terminal Targeting --> pCerulean_VP1up_NLS



    pCerulean_VP1up_NLS_Targeting molecule

    Investigator: Bea

    Comment: For preparing the next step in the VP1 insertion, different targeting molecules will be fused to the construct pCerulean_VP1up_NLS.

    1. The first construct is the Affibody ZEGF-R:1907 which was already cloned into the standard iGEM plasmid and can be sued for target tumor cells overexpressing the EGF receptor.
    2. The second construct is the mVenus (Yellow fluorescent protein) which can be used for imaging experiments
    3. Another motif will be the His-tag. Since the His-tag is very small, the oligos will be hybridized and directly ligated into the pCerulean backbone.


    Digestion of vector:

    • Plasmid used: pCerulean_VP1up_NLS (P365) c=470 ng/µL
    • Add BSA
    • AgeI-HF and SpeI-HF were used


    Protocol of the digestion of the vector:

    Components vpCerulean_VP1up_NLS/µL
    DNA 3
    BSA (100x) 2,5
    Buffer no. 4 (10x) 2,5
    Enzyme 1 AgeI 1
    Enzyme 2 SpeI HF 1
    H2O 15
    Total volume 25


    • Incubation of the sample at 37°C
    • Incubation for 120 minutes


    The Targeting molecules were digested with different enzmyes than the vector for providing the ability to assemble the different constructs together without creating new restriction site but to delete them. This works by fusing NgoMIv and AgeI together.

    Digestion of the targeting molecules

    • Plasmids used:
      1. pSB1C3_zEGF-R:1907 (P284) c=146 ng/µL
      2. pGA14_mVenus(P60) c=280 ng/µL
    • Add BSA
    • NgoMIV and SpeI-HF were used


    Components vZEGFR (P284)/µL vpGA_mVenus (P60)/µL
    DNA 13 µl 7 µl
    BSA (10x) 2,5 µl 2,5 µl
    Buffer no. 4 (10x) 2,5 µl 2,5 µl
    Enzyme 1 NgoMIV- 1,0 µl 1,0 µl
    Enzyme 2 AgeI HF 1,0 µl 1,0 µl
    H2O 15 µl 15 µl
    Total volume 25 25
    • Incubation of the sample at 37°C
    • Incubation for 90 minutes


    After incubation the samples were loaded on a 1% agarose gel. The obtained gel (after running the gel for 15 minutes) can be seen below.


    Results: The boxes mark fragments which ere cut out of the gel. The left lane (pCerulean) looks a bit strange, because Marker was added after the digestion reaction instead of Loading dye.

    Parallel, for assembling the His-Tag to the construct pCerulean_VP1up_NLS, oligos for the His-Tag in the RFC25 standard (provided by Gerrit) were hybridized and can directly be ligated in the digested vector. This strategy was used because of the smal size of the His-Tag which cannot be separated easily by the agarose gel.


    Hybridization of the His-Oligos

    • 10µL oligo1 (1:10)
    • 10µL oligo2 (1:10)
    • 4µL 100mM Tris-Cl pH 8
    • 10µL 5mM MgCl2
    • 8µL H2O

    The used programm for the hybridization:

    • 99°C 7´
    • 99°C 1´
    • -1°C R=0,3°/s
    • Goto 2 rep 74
    • Hold 4°C


    After the gel extraction of a T4 ligation was performed.

      Affibody ZEGF-R:1907:

      vCerulean_VP1up_NLS = 5,82µL
      vZEGF-R:1907 = 2,18µL

      mVenus Z:

      vCerulean_VP1up_NLS = 4,04µL
      vmVenus = 3,96 µL

      6xHisTag:

      vCerulean_VP1up_NLS = 0,2µL
      v6xHisTag = 7,98µL


    Next steps: Picking clones of the trafo plates (containing Kanamycin) and perform the Mini-Prep. The obtaining constructs finally will be fused to the VP2/3 protein which results in the final construct for the VP1 targeting approach.

    3rd Repetition of Mini-Preps and test digestion for Loop insertion BioBricks

    Investigator: Achim, Anna

    Sequencing results of Loop insertion BioBricks from 02.09.10; the following clones looked well:

    • pSB1C3_587_KO_RGD_clone6.4
    • PSB1C3_587_KO_His_clone9.4


    Update: 9 of the 14 ViralBricks looked well (sequencing results from 01. - 03.09.). Two new clones of the remaining constructs were prepared and test digested.

    Test digestion:

    components Volume for each sample /µl
    DNA 10
    BSA (10x) 1,5
    Buffer 4 (10x) 1,5
    Enzyme EcoRI HF 0,5
    Enzyme NotI HF 0,5
    H2O 1
    Total volume /µl15


    Components 453 BAP 453 Z34C 587 Z34C 587 KO Z34C 587 KO Z34C SPACER
    Clone1.5 + 1.6 11.5 + 11.6 12.5 + 12.6 13.5 + 13.6 14.5 + 14.6


    Incubation time: 1 h, Incubation temperature: 37°

    Preparation of gel:
    1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes

    3.9.10 AA.png


    Expected fragment sizes /bp:
    pSB1C3: 2051 bp
    BAP: ~150
    Z34C: ~ 200

    Comment: The following clones were sent for sequencing: 1.6, 12.6 ,13.6 and 14.5.
    To do: Retrafo of clone6.4 and clone9.4 and preparation of glycerol stocks.

    Cloning of Cap into pAAV_RC_ins-rep: preparation for SDM

    Investigator: Stefan

    Comment: Cloning Cap into pAAV still does not work as planned. As another approach Cap can be cut using BsiWI and XcmI and performing a SDM in the part of Cap not cloned into pAAV. Primers are ordered and should arrive on monday to proceed.


    Digestion:

    components pMA_RepCap Vector_SDM_InsPvuII (P211) pAAV_RC_ins-rep (P250)
    DNA 7,9 2,1
    Buffer 2 (10x)2 2
    XcmI 1 1
    BsiWI 11
    H2O8,1 13,9
    Total volume 20 20


    Comment:Digestion was performed using two steps, first incubating for 1 hour at 37 °C, afterwards for 1,5 hours at 55 °C.



    Gel:

    0,5 g Agarose,50 ml TAE (1%), 5 µl EtBr , at 115 Volt, running time: 50 minutes



    Freiburg10 pCerulean pMA pAAV.png




    Gelextraction:

    The gelextraction was performed according to the standard protocol. DNA concentration of the extracts:

    • pMA_RepCap Vector_SDM_InsPvuII (P211): c= 6,26 ng/µl
    • pAAV_RC_ins-rep (P250): c= 13,72 ng/µl


    Quick Ligation:

    Comment: XcmI produces only 1 base overhang, therefore Quickligase was used for ligation and incubation time increased.

    The Ligation was performed as following:

    • Vector Volume: 4,59 µl
    • Insert Volume: 4,41 µl


    • 10 µl QuickLigase buffer (2x)
    • 9 µl (Vector + Insert) mix
    • 1 µl QuickLigase


    Incubating for 60 minutes.


    Transformation:

    Trafo was performed according to the standard protocol (XL1b). The cells were plated on a agar plate with ampicilin.

    110. labday 04.09.2010

    Continuation: Cloning CFP_middlelinker (from pSB1C3_CFP_middlelinker, P276) into pCerulean (P273): Sequencing results of P381 and P385

    Investigator:Patrick

    Sequencing results of P381 & P385 Labelling: P381-CMV_rev & P385-CMV_rev: A wrong primer was used so there are no relevant sequence data yet. On sunday i will sent these clones again for sequencing. Primer: GATC_std_CMV-F

    Ha ich war schneller als Bea beim "Kopfkratzen-setzen" *lol*

    Hiermit verspreche ich dass es noch jede Menge Kopfkratzer, Laboraufsichtswichtel und pinke Panter geben wird (Patrick)









    Harvest viral particles, Transduction

    Investigator:Kerstin, Adrian

    • Harvest viral paricles from one plate of transfection from 01.09.2010 (10 plates, same treatment (10µg of each plasmid P357, P263, P356))
    • Transduction:
    1. 1x 6-well: 0,5ml of virus from 01.09.2010 (testing R/C P326)
    2. 1x 6-well: 0,5ml of virus from 01.09.2010 (testing R/C P325)
    3. 4x 6-well: 10x 0,5ml and 4x 1ml of virus from 01.09.2010 (stratagene R/C)
    4. 4x 6-well: 10x 0,5ml and 4x 1ml of virus from 04.09.2010 (10 plates same treatment)
    5. 6x 6-well: 0,5ml of virus from 21.08.2010 (TKGMK; 18x pH 7,10 and 6x pH 7,12)

    Repetition of Hybridisation and Cloning for Loop insertion BioBricks

    Investigator: Achim, Anna

    • Samples:

    1: 453_BAP
    11: 453_Z34C
    12: 587_Z34C
    13: 587_KO_Z34C
    14: 587_KO_Z34C_Spacer


    Comment: New approaches of the Hybridisation and Fill-in reactions were done because it didn't work the first time. A possible reason is that the klenow enzyme wasn't inactivated. In addition, the conditions for ligation will be improved. Also the ligation of 453_BAP was done again.

    Digestion:

    components Volume Vector 587 Volume Vector 453 Sample 11 Sample 12 Sample 13 Sample 14
    DNA 3,7 3,7 6 6 6 6
    BSA (10x) - - - - - -
    Buffer 4 (10x) 2 2 2 2 2 2
    Enzyme 1° Bam Ssp Ssp Bam Bam Bam
    Enzyme 2° Pvu Sal Sal Pvu Pvu Pvu
    H2O 12,3 12,3 10 10 10 10
    Total volume /µl20


    ° 1 µl each

    Preparation of gel:
    1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 50 minutes

    Repetition: Prepatation for SDM: cloning of Cap into pAAV

    Investigator: Stefan

    Schönen Abend dir noch

    Comment: On yesterday's plates nothing grew. Because gelex and ligation products were stored in coldroom, new ligation and trafo approaches were performed.



    Ligation approaches:

    • ligation with Quick Ligase (as performed yesterday)
    • ligation with T4 Ligase



    Transformation approaches:

    • Quick Ligase (approach from yesterday):
      • 2 µl of ligation product
      • 4 µl of ligation product


    • Quick Ligase (new approach):
      • 2 µl of ligation product
      • 4 µl of ligation product


    • T4 Ligase:
      • 2 µl of ligation product
      • 4 µl of ligation product


    Transformation:

    Trafo of each approach was performed according to the standard protocol (XL1b). The cells were plated on a agar plate with ampicilin.

    Ligation and trafo of the modified Cap insert , the Viral Bricks 1, 11, 12, 13 and 14

    Investigator: Volker


    Ligation


    Construct Vector (µl) Insert(µl)
    ViralBrick 1 in 453 7.91 0.09
    ViralBrick 11 in 453 6.17 1.83
    ViralBrick 12 in 587 6.15 1.85
    ViralBrick 13 in 587 5.3 2.7
    ViralBrick 14 in 587 4.46 3.54
    p211 in pAAV-RC 5.52 3.54
    p211 in pAAV-RC 5.52 3.54



    Preparation of PBS Buffer

    Investigator: Volker

    Two liters of PBS buffer were prepared for the column purification of the viral particles. The following protocol was used:

    • dissolve the following in 800ml ddH2O:
      • 8g of NaCl
      • 0.2g of KCl
      • 1.44g of Na2HP0$ (1.69 because of the crystal water in the reagent)
      • 0.24 g of KH2PO4
    • adjust to pH 7.4
    • adjust to 1000ml
    • steril filtrate the solution

    111. labday 05.09.2010

    Mini-prep of glycerol stock pMA_RepCap Vector_SDM_InsPvuII clone 1 (B182)

    Investigator: Stefan

    Glycerol stock: Because glycerol stock B182 was not mixed before putting into -80 °C freezer the cells died. Therefore a new glycerol stock was prepared and labled B302.

    Mini-Prep was performed according to the standard protocol. Two preps were prepared:

  • P363 = pMA_RepCap Vector_SDM_InsPvuII clone 1 (1) = 215,6 ng/µl
  • P364 = pMA_RepCap Vector_SDM_InsPvuII clone 1 (2) = 230,5 ng/µl

    Picking clones of pCerulean_Zegfr:1907_"Linker" and pCerulean_CFP_MiddleLinker

    Investigator: Hanna

    2 clones of each construct (1. pCerulean_Zegfr:1907_ShortLinker, 2. pCerulean_Zegfr:1907_MiddleLinker, 3. pCerulean_Zegfr:1907_LongLinker, 4. pCerulean_Zegfr:1907_SEG and 5. pCerulean_CFP_MiddleLinker) were picked.

    To do tomorrow (6.9.): Mini-Preps and Test digestion (with EcoRI and PstI).


    112. labday 06.09.2010

    Minipreps & Test PCR of pTert Biobrick assembly

    Investigator: Achim

    • After unsucessful test digestion with EcoRI and PstI I tried out a test PCR with Igem Primers (same Mastermix as for loop inserts)
    • Elongation time was set to 2.35 min because the amplified fragment should be about 2.5 kbp big.
    • Result:
    The visible bands are not specific. Either cloning or PCR went wrong :)
    • Next steps: Another test digestion tomorrow, if that doesn't work out I'll repeat the ligation.

    Minipreps & Test PCR of Loop inserts 1, 11, 12 ,13, 14

    Investigator: Achim & Anna

    • Samples:

    1: 453_BAP
    11: 453_Z34C
    12: 587_Z34C
    13: 587_KO_Z34C
    14: 587_KO_Z34C_Spacer


    • Minipreps of 1.1, 1.3, 11.1, 11.2, 12.1, 12.2, 13.1, 13.2, 14.1, 14.2
    • Clones 1.4, 11.3, 11.4, 12.3, 12.4, 13.3, 13.4, 14.3, 14.4 were pelleted and stored away in case the first two are incorrect.
    • In the hope to confirm our small inserts, A test PCR using Igem standard primers was carried out:

    PCR-reaction:

    Volume / µl ingredients
    2,5 Taq Buffer
    x template (~0.5 ng)
    2,5forward Igem primer (1:10)
    2,5 reverse Igem primer (1:10)
    0,5dNTP
    filled upt to 25H2O
    0,5Taq Polymerase (1.25)


    PCR program:

    Roundstemperature/ °CTime
    1 95 30"
    9525"
    25 61 30"
    68 50"
    1 68 5"


    Results:

    Bands should be about 150 for BAP and about 200 for Z34C. Bands dont quite match the expectations, somehow the BAP band seems bigger than the Z34C band. Clones 1.3, 11.1, 12.2, 14.1 were sent for sequencing.



    Mini-Preps and test digestion of Linker BioBricks

    Investigator: Anna

    Comment: Mini-preps of all clones were prepared. They have not been added to the excel sheets yet, but were stored in the freezer.

    Samples 1 2 3 4 5 6
    InsertVP1up_NLS_HisVP1up_NLS_His ZeGFP:1907_SEG ZeGFP:1907_SEG ZeGFP:1907_ML ZeGFP:1907_ML
    Clone12 1 2 1 2
    Concentration439,57463,51 415,24 354,5 406,75 382,27


    7 8 9 10 11 12
    CFP_ML CFP_MLZeGFP:1907_SLZeGFP:1907_SLZeGFP:1907_LLZeGFP:1907_LL
    1 2 1 2 1 2
    482,76424,76 497,06 413,55 391,05427,20

    SL: Shortlinker ML: Middlelinker LL: Longlinker

    Test digestion:

    components Volume for each sample /µl
    DNA 1
    BSA (10x) -
    Buffer 4 (10x) 1
    Enzyme EcoRI HF 0,5
    Enzyme Pst HF 0,5
    H2O 7
    Total volume /µl 10


    Preparation of gel:

    • Incubation time: 1 h, Incubation temperature: 37°
    • 1 g Agarose, 100 ml TAE (1%), 6 µl GELRED , at 115 Volt, running time: 45 minutes



    Freiburg10 pCerulean Linker 06.09.png

    • Expected fragment sizes:

    VP1up_NLS_Histag: 550 bp
    ZeGFP:1907_SEG: 344 bp
    ZeGFP:1907_ML: 261 bp
    CFP_ML: ~ 1000 bp
    ZeGFP:1907_SL: 249 bp
    ZeGFP:1907_LL: 237 bp

    To do: Test digestion of VP1up_NLS_mVenus and Affibody. Number 2, 3, 5, 7, 9 and 12 will be sent for sequencing tomorrow. For sequencing results of VP1up_NLS_Histag go to labday 09.09.2010, for the linker biobricks go to labday 08.09.2010.

    Production of Ampicilin and Chloramphenicol

    Investigator: Jessica

    • 10ml ethanol (70%) containing 1g Amp in 60µl aliquots
    • 10ml ethanol (70%) containing 0,25g Cm in 60 µl aliquots


    Mini-prep and test digestion of pAAV_RC-CapIns_prepSDM

    Investigator: Stefan

    Mini-Prep was performed according to the standard protocol

  • P394 = pAAV_RC_CapIns_prepSDM clone 1 = 296,42 ng/µl
  • P395 = pAAV_RC_CapIns_prepSDM clone 2 = 274,92 ng/µl
  • P396 = pAAV_RC_CapIns_prepSDM clone 3 = 498,39 ng/µl
  • P397 = pAAV_RC_CapIns_prepSDM clone 4 = 487,67 ng/µl
  • P398 = pAAV_RC_CapIns_prepSDM clone 5 = 469,96 ng/µl
  • P399 = pAAV_RC_CapIns_prepSDM clone 6 = 517,06 ng/µl
  • P400 = pAAV_RC_CapIns_prepSDM clone 7 = 451,68 ng/µl
  • P401 = pAAV_RC_CapIns_prepSDM clone 8 = 479,18 ng/µl
    Constructs were digested with XcmI and Acc65I for 70 minutes at 37°C
    components pAAV_RC_CapIns_prepSDM clone 1-8
    DNA 2
    Buffer 2 (10x)1
    BSA (10x)1
    XcmI 0,5
    Acc65I 0,5
    H2O5
    Total volume 10



    Freiburg10 pAAV RC CapIns prepSDM.png


    Sent for sequencing: Clone 3 (P396) was sent for sequencing.

    • Tube name: SB1
    • Primer used: VP 4200 rev

    Sent for sequencing

    Investigator: Stefan

    • Tube name: SB2

    pAAV_RC_inserts clone1 (P326) was sent for sequencing using the following primers:

    • VP1 primer for pKex rev
    • SK
    • Primer Cap 2800 for
    • Primer Cap 2800 rev
    • Primer Cap 3500 for