Team:Freiburg Bioware/testpage

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Contents

18. Labortag 01.06.2010: Modifying MCS of pAAV_MCS vector


Investigators: Anissa, Adrian, Bea, Chris W., Hanna, Patrick, Volker, Sven

Oligos received from Sigma-Aldrich
(right ITR of pAAV_MCS, left ITR of pAAV_MCS and MCS RFC25 for pAAV)

  • Hybrization of received oligos: MCS RFC25 for pAAV (forward) and MCS RFC25 for pAAV (reverse)
  • Centrifuge tubes prior to open tubes (13.000 rpm, 30 sec)
    • MCS RFC25 for pAAV (forward): Add 92µL Millipore H2</sup>O (Volume on obtained sheet)
    • MCS RFC25 for pAAV (reverse): Add 394 µL Millipore H2</sup>O (Volume on obtained sheet)
  • Vortex the resuspended DNA
  • Make aliquots of both Oligos (1:10): 10 µL Oligo + 90 µLH2</sup>O (final volume usually 100 µl)
  • Mix together(into PCR-tube):


Volume/µL solution
10 (1:10)Oligo 1: MCS RFC25 for pAAV (forward)
10Oligo 2: MCS RFC25 for pAAV (reverse)
4 100mM TrisHCl pH8
85mM MgCl2
8 H20


  • Program: ORIGAMI 1 modified for long oligos:
    • 1 99°C 7’
    • 2 99°C 1’
    • -1°C R=0.3 °/s
    • Goto 2 rep 74
    • Hold 4°C


  • While hybridization of oligos is performed, digestion of pAAV_MCS vector can be conducted

following standard protocol for cloning.

  • Title: Ligation MCS_Oligo with pAAV_MCS
  • Plasmid: pAAV_MCS
  • Buffer used: 3
  • BSA: Yes
  • Measure DNA-concentration with Nanodrop
  • DNA-Concentration:260 ng/uL
  • Restriction-enzyms used: http://www.neb.com/nebecomm/DoubleDigestCalculator.asp

Enzyme1 (Nr. Lab: 152): ClaI Enzyme2 (Nr. Lab: 15): BglII

  • Digestion components :
components pAAV_MCS
DNA 4
BSA (10x) 3
Buffer 3 (10x)3
Enzyme: ClaI (no.Lab:152)2
Enzyme: BglII (no.Lab:15)1
H2</sup>O17
Total volume30
  • Incubate for 1,5 h at 37°C


  • 1% Agarose gel
    • 1% agarose gel was prepared, gel ran for 45 minutes( first: 90V, after 15 minutes: 115 V)


  • Amount of loading dye added
sample/µL loading dye/µL
marker: 8 contains loading dye
pAAV_MCS: 24 6


  • Expected size of fragments
sample expected size
pAAV_MCS: cut with ClaI and BglII4580 bp


19. Labortag 02.06.2010: Oligos (NotI)

Investigators: Adrian, Bea, Chris W., Hanna, Anissa

Practical work:
Control plate contained no clones. :) 4 colonies were picked and grown @ 37°C over night.
Theoretical work:
Oligos for site directed mutagenesis of the NotI restriction sites in pAAV_MCS (ITRs) were designed:File:Freiburg10 NotI ITR Oligos.pdf
Sponsoring work:
Sponsoring letter was adapted for Quiagen.

20. Labortag 03.06.2010: pAAV_RFC25_MCS -> problem

Investigators: Anissa, Bea, Melanie, Christian L.

Comment: Continue with Mini-Prep and test digestion of pAAV_RFC25_MCS
Mini-Prep and test digestion have been performed:

Problem: Designed oligos (MCS_RFC25) for altering the MCS of the pAAV_MCS vector cannot be used.
Two startcodons are in the MCS.

Oligo MCS_RFC25: contains 2 Startcodons. the "wrong" Codon is the first ATG which results in peptide chain with 30 aa. The second ATG is the right ATG which is right before the Gene of Interest.










The two startcodons are not in the same open reading frame (ORF). Therefore two proteins will be produced. The Gene of Interest and the short peptide (30 aa).
The idea of the oligos was to ligate the oligos into the pAAV_MCS vector. The oligos contained two overhangs which correspond to the sequences of the two restriction sites ClaI and BglII:
The pAAV_MCS vector was digested with ClaI and BglII and then we ligated the oligo and the vector. Problem was that we did not notice that the overhang of ClaI and the sequence of EcoRI of the MCS_RFC25 resulted in another ATG startcodon.

Possible Solutions:

    1. first: modify MCS with ordered oligos of Sven (shorter MCS which cannot be used for pEX)and clone mVenus
    2. Perform site-directed-mutagenesis (QuikChange from Stratagene)
    3. Order new MCS-oligos and consider that no new ATG is produced. For example: add another base between ClaI overhang and EcoRI sequence. -----ATXG---- This solution is the more possible one we are going to perform.


Practical work

    • Preparing four glycerol stocks (2:1)
      • numbers: B4 - B7 (for details see nomenclature)
      • stored in -80°C, Box 1
  • MiniPrep
    • Nanodrop concentrations


Sample Concentration/ng*µl-1
P11 340,5
P12 364,0
P13 358,5
P14 284,4


  • Test digestion


Components Volume µl Mastermix µl
DNA 800 --
BSA (10x) 1,5 7,5
Buffer No.2 (10x) 1,5 7,5
Enzyme 1 (no.Lab:45) Nde I 0,5 2,5
Enzyme 2 (no.Lab:71) Spe I 0,5 2,5
H2</sup>O variable --
Total volume 15 20


Sample Volume/ µl H2</sup>O / µl
P11 2,3 8,7
P12 2,2 8,8
P13 2,2 8,8
P14 2,8 8,2
  • Incubation: 1,5 h


  • Agarose-Gel
    • Materials
    0,5 g Agarose, 50 ml TAE, 3µl GELRED, at 100 Volt, running time: 45 minutes
    Sample Sample/µl] Loading dye (5x/6x)/µl Expected size 1 (Geneious) Expected size 2 (Geneious)
    P11 15 µl 3 µl 3677 bp 974 bp
    P12 15 µl 3 µl 3677 bp 974 bp
    P13 15 µl 4 µl 3677 bp 974 bp
    P14 15 µl 4 µl 3677 bp 974 bp


    Marker Sample P11 /18 µl Sample P12 /18 µl Sample P13 /19 µl Sample P14 /19 µl
    Lane 1 3 5 7 9

    Results of agarose-gel:

      • Expected fragments of 3677 bp and 974 bp can be cerified. The insertion of the RFC25_MCS has been inserted.
      For further verification the insert has to be sequenced has to be conducted (to do for 04.06.2010).


    Picking clones of Thymindinkinase of Amor

      • 5 clones of the XL-1 Blue colonies containing the plasmid pUB6/HV5/His6 with the thymidinkinase have been picked from LBamp-agarplates
      • 1 clone of the XL-1 Blue colonies containing the plasmid pUB6/HV5/His6 without the thymidinkinase (control) has been picked from LBamp-agarplates
      • all clones have been inoculated in 10 mL LB containing 10 µL Amp. Incubation: 37°C over-night.
      • to do: Mini-Prep of pUB6/HV5/His6 with the thymidinkinase and pUB6/HV5/His6 without the thymidinkinase (control)


    Idea

      • Insertion of Kozak consensus sequence before MCS to enhance gene expression (cloning consideration in Stratagene manual)
        • New RFC standard with Kozak sequence for eucaryotes??

      21. Labortag 04.06.2010: DKFZ plasmid Retrafos, TK/GMK Mini-Prep

      Investigators: Adrian, Bea, Chris W., Hanna

      DKFZ

      Comments: Plasmids of PD Kleinschmidt of the DKFZ arrived. The DNA was dried on a whatman paper.
      Plasmids received:

      • pXX6 alle Adenovirus Helfer Gene ohne AAV Gene; Plasmid von J. Samulski; evtl. Anfragen für Benutzererlaubnis
      • pKEX-2XL.Rep40 Expressionsplasmide für das Rep Proteine 40
      • pKEX-2XL.Rep 52 Expressionsplasmide für das Rep Proteine 52
      • pKEX-2XL.Rep 68 Expressionsplasmide für das Rep Proteine 68
      • pKEX-2XL.Rep 78 Expressionsplasmide für das Rep Proteine 78
      • pCMV-VP(HS) Expressionsplasmid der drei VP Proteine in Kombination (assemly kompetent
      • pKEX-VP1 Expressionsplasmide für die einzelnen VP (cap) Proteine (VP1, VP2 und VP3) vorsicht: sind alleine nicht assembly kompetent; Ruffing et al 1992; Steinbach et al. 1997;
      • pKEX-VP2Expressionsplasmide für die einzelnen VP (cap) Proteine (VP1, VP2 und VP3) vorsicht: sind alleine nicht assembly kompetent; Ruffing et al 1992; Steinbach et al. 1997;
      • pKEX-VP3Expressionsplasmide für die einzelnen VP (cap) Proteine (VP1, VP2 und VP3) vorsicht: sind alleine nicht assembly kompetent; Ruffing et al 1992; Steinbach et al. 1997;
      • pTRUF_CMV_eGFPEinzelstrang Vektor zur Expression von eGFP
      • dsAAV_CMV_eGFP"self complementary" Expressionsvektor von X. Xiao; evtl. anfragen wegen Benutzererlaubnis
      • pTAV2 (gesamtes AAV Genom im "blue script" Vektor)
      • pDG komplettes Helferplasmid zur Vektorherstellung; Grimm et al. 1998


      • In order to obtain the DNA following steps have been performed:
        • cut out the spot where the DNA is spotted with a clean scalpel (note: scalpel should not have any contamination).
        • put a 0,5 mL Eppi in a 1,5 mL Eppi. Put little holes in the smaller eppi.
        • transfer Whatman paper into Eppi
        • add 50 µL TE-EF (Redissolving buffer) to whatman paper and wait 15 minutes
        • centrifuge eppis at 2000 rpm, 10 minutes
      • Transformation with obtained plasmids was performed.


    Fusion Enzyme: Thymidinkinase/Guanylate Kinase (TK/GMK)

    Plasmid Mini-Prep

    • experiment date: 04.06.2010 ; time: 3,5h
    • name of investigator: Adrian, Bea, ChrisW.,Hanna
    • new vector name: pUB_V5_His6 + TK/GMK (fusionenzyme)


    Glycerol Stocks

    Clone 1 Clone 2 Clone 3 Clone 4 Clone 5Control
    Bacteria strain XL-1 Blue XL-1 Blue XL-1 Blue XL-1 Blue XL-1 Blue XL-1 Blue
    Plasmidname pUB_V5_His6 + TK/GMK pUB_V5_His6 + TK/GMK pUB_V5_His6 + TK/GMK pUB_V5_His6 + TK/GMK pUB_V5_His6 + TK/GMK pUB_V5_His6 + TK/GMK
    Date 04.06.2010 04.06.2010 04.06.2010 04.06.2010 04.06.2010 04.06.2010
    given number B8 B9 B10 B11 B12 B13


    Given Plasmid-Number

    Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Control
    given number P15 P16 P17 P18 P19 P20


    Nanodrop concentration

    • Plasmid
      • Given Plasmid-Number: P15; DNA concentration: 493,7 ng/µL ;
      • Given Plasmid-Number: P16; DNA concentration: 464,4 ng/µL;
      • Given Plasmid-Number: P17; DNA concentration: 445,6 ng/µL;
      • Given Plasmid-Number: P18; DNA concentration: 562,9 ng/µL;
      • Given Plasmid-Number: P19; DNA concentration: 528,1 ng/µL;
      • Given Plasmid-Number: P20; DNA concentration: 499,9 ng/µL;


    Comments:A Plasmid-Mini Prep with the received Fusionenzyme Thymidinkinase/Guanylate Kinase (TK/GMK)from Amor has been performed.
    The DNA will be sent to GATC for sequencing.

    • pUB6_V5_His6 - clone 1 (P15) (3 µL added to 27µL H20) has been sent to GATC.
    • Expected results: Saturday



    22. Labortag 05.06.2010: Insertion of iGEM expression parts into pAAV_MCS

    Investigators: Adrian, Bea, Melanie, Hanna

    Hybridization:

    • Hybridization of received oligos: iGEM expression parts (RFC25 without EcoRI, NotI)
    • Prior to opening the tubes, they were centrifugated at 13.000 rpm for 30 sec.
      • expression part MCS_for (charge-no: ST00114065): 108 µL Millipore H2</sup>O (Volume on obtained sheet)were added.
      • expression part MCS_for (charge-no: ST00114066): 165 µL Millipore H2</sup>O (Volume on obtained sheet)were added.
    • Resuspended DNA was vortexted.
    • Aliquots of both Oligos (1:10) were prepared: 10 µL Oligo + 90 µL H2</sup>O (final volume usually 100 µl).
    • Mix together(into PCR-tube):


    Volume/µL solution
    10 (1:10)Oligo 1: expression part MCS_for (charge-no: ST00114065)
    10 (1:10)Oligo 2: expression part MCS_for (charge-no: ST00114066)
    4 100 mM TrisHCl pH8
    85 mM MgCl2
    8 H20


    • Program: ORIGAMI 1 modified for long oligos:
      • 1 99°C 7’
      • 2 99°C 1’
      • -1°C R=0.3 °/s
      • Goto 2 rep 74
      • Hold 4°C


    • While hybridization of oligos was performed, digestion of pAAV_MCS vector was conducted

    following standard protocol for cloning.

    Digestion:

    • Title: Ligation iGEM expression parts (="iGEM-MCS") with pAAV_MCS
    • Plasmid: pAAV_MCS
    • Buffer used: 3
    • BSA: Yes
    • DNA-Concentration: 260 ng/uL
    • Restriction-enzyms used:

    Enzyme1 (Nr. Lab: 152): ClaI Enzyme2 (Nr. Lab: 15): BglII

    • Digestion components :
    components pAAV_MCS
    DNA 5.8 µL
    BSA (10x) 3 µL
    Buffer 3 (10x)2 µL
    Enzyme: ClaI (no.Lab:152)2 µL
    Enzyme: BglII (no.Lab:15)1 µL
    H2</sup>O16.2 µL
    Total volume30 µL
    • Mixture was incubated for 1,5 h at 37°C.


    Agarose-Gel:

    • 1% agarose gel was prepared, gel ran for 45 minutes(110 V)


    • Amount of loading dye added
    sample/µL loading dye/µL
    marker: 8 contains loading dye
    pAAV_MCS: 30 6 (6x loading dye)


    • Expected size of fragments
    sample expected size
    pAAV_MCS: cut with ClaI and BglII4580 bp


    </ul>

    Gelextraction:


    Gel measurement:

    Sample weight
    pAAV_MCS 60 mg
    • Gelextraction was performed following standard protocol.
    • DNA-concentrations were meassured: pAAV_MCS 18.2 ng/µL, Oligos: 136.7 ng/µL -> 1:10 dilution was prepared: 13.67 ng/µL



    Ligation:


    iGEM-MCS pAAV_MCS
    Volume/µl 0.4 8.6


    Trafo was performed (using XL1B cells) following standard protocol.

    23. Labortag 07.06.2010: Mini-Preps (Kleinschmidt-plasmids and pAAV_iGEM-MCS)

    investigators: Achim, Kira,Jessy, Chris W., Hanna, Adrian, Bea

    pAAV_iGEM-MCS

    Plasmid Mini-Prep

    • experiment date:07.06.2010; time: whole day
    • name of investigator: Achim, Kira,Jessy, Chris W., Hanna, Adrian, Bea


    Glycerol Stocks

    Clone 1 Clone 2 Clone 3 Clone 4
    Bacteria strain XL1B XL1B XL1B XL1B
    Plasmidname pAAV_iGEM-MCS pAAV_iGEM-MCS pAAV_iGEM-MCS pAAV_iGEM-MCS
    Date 07.06.2010 07.06.2010 07.06.2010 07.06.2010
    given number - B27 B28 B29


    Given Plasmid-Number

    Clone 1 Clone 2 Clone 3 Clone 4
    given number - P34.2 P34.3 P34.4


    Test digestion

    • buffer used: 4 ; Restriction-enzymes used: Enzyme 1 (no. Lab:___) AgeI ; Enzyme 2 (no.Lab:___) NdeI
    • Plasmid
      • Given Plasmid-Number: P34.2; DNA concentration: 433.78 ng/µL ;
      • Given Plasmid-Number: P34.3; DNA concentration: 408.48 ng/µL ;
      • Given Plasmid-Number: P34.4; DNA concentration: 409.80 ng/µL ;


    Comments:Clone no.1 was dismissed...

    Test digestion:

    Components Volume/µL Mastermix
    DNA (clone 2) 1000 ng -
    BSA (10x) no -
    Buffer no. 4 (10x) 1.5 µL -
    Enzyme 1 (no. Lab: ) AgeI 0.75 µL -
    Enzyme 2 (no. Lab: ) NdeI 0.5 µL -
    H2</sup>O variable -
    Total volume 15 µL -


    Sample Volume sample/ µl Volume H2</sup>O / µl
    P34.2 2.3 9.95
    P34.3 2.4 9.85
    P34.4 2.4 9.85
    • Incubation: 45 min, 37°C


    Agarose-Gel:


    0.5 g Agarose, 50 ml TAE (1%), 3 µL GELRED (3-6µl), at 110 Volt, running time: 45 minutes

    Sample Sample/µl] Loading dye (5x/6x)/µl Expected size 1 (Geneious) Expected size 2 (Geneious)
    P34.2 15 µl 3 µl 3686 bp 951 bp
    P34.3 15 µl 3 µl 3686 bp 951 bp
    P34.4 15 µl 3 µl 3686 bp 951 bp


    • Marker: GeneRuler ladder mix
    Marker Sample Sample Sample
    Lane 34.2 / 18 µl 34.3 / 18 µl 34.4 / 18 µl



    Kleinschmidt-plasmids

    Plasmid Mini-Prep

    • experiment date: 07.06.2010 ; time: 10 – 20 h
    • name of investigator: Kira, Achim, Jessy, Bea, Adrian, Hanna
    • Kleinschmidt-plasmids


    Glycerol Stocks

    Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 Clone 11 Clone 12 Clone 13
    Bacteria strain XL1B XL1B XL1B XL1B XL1B XL1B XL1B XL1B XL1B XL1B XL1B XL1B XL1B
    Plasmidname pXX6 pKEX-2XL.Rep 40 pKEX-2XL.Rep 52 pKEX-2XL.Rep 68 pKEX-2XL.Rep 78 pCMV-VP(HS) pKEX-VP1 pKEX-VP2 pKEX-VP3 pTRUF_CMV_eGFP dsAAV_CMV_eGFP pTAV2 pDG
    Date 07.06.2010 07.06.2010 07.06.2010 07.06.2010 07.06.2010 07.06.2010 07.06.2010 07.06.2010 07.06.2010 07.06.2010 07.06.2010 07.06.2010 07.06.2010
    given number B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26


    Given Plasmid-Number

    Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 Clone 11 Clone 12 Clone 13
    given number P21 P22 P23 P24 P25 P26 P27 P28 P29 P30 P31 P32 P33
    measured concentration 351,01 673,1 532,22 579,05 725,31 659,68 692,8 545,46 568,34 420,62 446,95 496,8 472,58



    Comments: Many things went wrong today!

    • Glycerol stocks must be vortexted!
    • Check mini-prep buffers - especially buffer PE (needs to contain ethanol!)
    • Always check volumes - try to estimate if volume makes sense (check pipettes!)
    • Don't discard bacteria cultures until glycerol stocks and mini-preps are successfully done!!!

    Today's conclusion: Better ask 2 times than do something wrong without asking!!!


    Site-directed mutagenesis of pAAV_iGEM-MCS (PstI)


    Quickchange site directed mutagenesis: PCR reaction:

    • 2.5 µL 10x Pfu Ultra II buffer
    • 0.5 µL template (therefore the a 1:20 dilution of the pAAV_iGEM-MCS (433 ng/µL) was prepared) = 10.825 ng
    • 0.56 µL primer 1 (of 1:10 dilution)
    • 0.56 µL primer 2 (of 1:10 dilution)
    • 1 µL DMSO (primers form very strong secondary structures)
    • 0.5 µL dNTP
    • 18.88 µL dH2</sup>O
    • 0.5 µL PfuUltra II fusion (1.25 U)

    -> end volume: 25 µL

    PCR program:
    1 x : 2' 95°C (HotStart polymerase) 20 x : 30 s 95°C -> 1' 55°C -> 5' 68°C 1 x : 4°C (over night)
    Experiment will be continued tomorrow.

    24. Labortag 08.06.2010: Cloning of mVenus_YFP into pAAV_iGEM-MCS, continuation of site-directed mutagenesis

    Investigators: Kira, Jessy, Hanna, Achim

    Analysis of iGEM-MCS sequence (RFC25 without EcoRI and NotI):


    Freiburg10 iGEM-MCS.jpg
    The alignment with the theoretical pAAV_iGEM-MCS delivered a "C-deletion" within the NgoMIV restriction site. Due to that the pAAV_iGEM-MCS lacks this site. We controlled the bill of delivery and noted that the deletion must be GATC's fault. All in all this doesn't matter, because parts which are in iGEM standard can be inserted and therefore they will deliver the lacking NgoMIV restriction site. Further on this could be an advantage because after digestion the fragment which is cut out can be better detected in the gel.

    Insertion of mVenus_YFP into pAAV_iGEM-MCS

    Digestion:

  • plasmid: insert: pGA14mVenusGeneart; number: - origin:Sven
  • plasmid: vector: pAAV_iGEM-MCS; number: P32.2 production date:05.06.2010 origin: ____
  • new vector name: pAAV_iGEM-MCS_mVenus
  • buffer used:4 ; Restriction-enzymes used: Enzyme AgeI (no. Lab:149); Enzyme XbaI (no. Lab: 63)
  • DNA concentration (vector):433,78ng/µl ; DNA concentration (insert): 530 ng/µl

    components V (pAAV_iGEM-MCS)/ µl I(pGA14mVenus_Geneart) / µl
    DNA 2,33,8
    BSA (10x) 22
    Buffer 3 (10x)22
    Enzyme: AgeI (no.Lab:149)1,251,25
    Enzyme: XbaI (no.Lab:63)0,750,75
    H2</sup>O11,710,2
    Total volume2020
  • Incubation: 1 1/2 h at 37°C
    0.5 g Agarose, 50 ml TAE (1%), 3 µL GELRED (3-6µl), at 115 Volt, running time:

    Sample Sample/µl] Loading dye (6x)/µl Expected size 1 (Geneious) Expected size 2 (Geneious)
    P34.2 20 µl 4 µl 4617 bp 22 bp
    pGA14mVenus 20 µl 4 µl 2870 bp 774 bp



  • Gel loaded with vector and insert samples, 24 µl each & 8 µl marker
  • after 20 minutes, the insert band was cut out. Two overlapping bands were visible in the vector well, after 1 1/2 hours those bands were separated and both were cut out.

    Sample weight concentration
    Vektor_Oben 0,32 g 29,8 ng/µl
    Vektor_Unten 0,14 g 12,8 ng/µl
    Insert0,3 g 12,9 ng/µl



  • the exact volume of insert and vector was calculated with LabTools:
  • Ligation I with Vektor_Oben:
      • Vector: 4,16 µl
      • Insert: 4,84 µl
  • Ligation II with Vektor_Unten:
      • Vector: 6 µl
      • Insert: 3 µl


    continuation of site-directed mutagenesis

    Digestion with DpnI:

  • 0.5 µl DpnI added
  • incubated for 1 hour at 37°C

    Trafo:

  • Cells used for transformation: XL1B
  • Centrifugation for 3 min at 8000 rpm instead of 6000 rpm (accidentaly)
  • Plated on agar plate: pAAV_IGEM-MCS Mutagenese PST1 8.6.2010 AM XL1B
  • incubated over night

    25. Labortag 09.06.2010: pAAV_iGEM-MCS_mVenus-YFP, site-directed mutagenesis

    investigators: Jessy, Achim, Sven, Toby, Hanna

    No pAAV_iGEM-MCS_w/oPstI transformed bacteria (site-directed mutagensis) grew on the ampicillin agar plates over night!

    Experiment is conducted again by Toby (Thanks a lot!!!).


    Further on the cloning of mVenus-YFP into pAAV_iGEM-MCS seemed to fail:

    Only on the agar plate cotaining the bacteria transformed with the "vector_oben" ligation, which actually should be the "wrong" ligation (gelextraction of a band which contained fragments that were too large, see picture in lab journal), revealed colonies. Therefore this experiments is also conducted one more time by Sven (thanks also a lot!!!)


    Insertion of mVenus_YFP into pAAV_iGEM-MCS

    • experiment date: 9.6.2010
    • name of investigator: Sven
    • plasmid:
      • Vector: name: pAAV_iGEM-MCS number: 34.2 production date: 5.6.2010
      • Insert: name: pOG14_mVenus number: - production date: ____ origin: Sven :)
    • new vector name: pAAV_iGEM-MCS_mVenus-YFP
    • buffer used: NEB4 ; Restriction-enzymes used: Enzyme 1 (no. Lab:___) AgeI ; Enzyme 2 (no.Lab:___) XbaI
    • DNA concentration (vector): 433 ng/µL ; DNA concentration (insert): 530 ng/µL



    Digestion

    components volume of vector /µl volume of insert /µl
    DNA 2.83 2.77
    BSA (100x) 0.5 0.5
    Buffer NEB4 (10x) 3.0 3.0
    Enzyme 1 (AgeI) 1.75 1.75
    Enzyme 2 (XbaI) 1.25 1.25
    H2</sup>O20.73 20.67
    Total volume (e.g. 15,20,25,30 µl) 30 30



    Agarose-Gel:


    0.5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED (3-6µl), at 115 Volt

    Gelextraction


    • insert (mVenus-YFP): 26 ng/µL
    • vector (pAAV_iGEM-MCS): 30 ng/µL

    Ligation


    • vector: 5.85 µL
    • insert: 3.15 µL
    • ligase: 1 µL
    • buffer: 10 µL



    Test digestion of pAAV_iGEM-MCS

    p style="font-size:15px; font-weight: bold; color: blue;">Test digestion</p>

    • buffer used: 1 ; Restriction-enzymes used: Enzyme 1 (no. Lab:___) NgoMIV
    • Plasmid
      • Given Plasmid-Number: P34.2 ; DNA concentration: 433.78 ng/µL;
      • Given Plasmid-Number: P34.3 ; DNA concentration: 408.48 ng/µL;
      • Given Plasmid-Number: P34.4 ; DNA concentration: 409.8 ng/µL;



    Components P34.2 [µL] P34.3 [µL] P34.4 [µL]
    DNA 2.3 2.5 2.4
    BSA (10x) 1 1 1
    Buffer no. 1 (10x) 1 1 1
    Enzyme 1 (no. Lab: 113) ngoMIV 1 1 1
    H2</sup>O 4.7 4.5 4.6
    Total volume 10 10 10
    • Incubation: 1 h, 37°C


    Agarose-Gel:


    0.5 g Agarose, 50 ml TAE (1 %), 3 µL GELRED, at 115 Volt, running time: 45 minutes + 20 minutes

    Sample Sample/µl] Loading dye (6x)/µl Expected size 1 (Geneious) Expected size 2 (Geneious)
    P34.2 10 µl 2 µl 3728 bp 903 bp
    P34.3 10 µl 2 µl 3782 bp 903 bp
    P34.4 10 µl 2 µl 3782 bp 903 bp


    • Marker: GeneRuler ladder mix
    Marker Sample P34.2 /µl Sample P34.3 /µl Sample P34.4 /µl
    Lane 8 µL 10 µL 10 µL 10 µL


    Comments: Digestion of P34.2 delivered just one fragment size, revealing that - as expected after sequencing (C-deletion: no NgoMIV site in iGEM-MCS) - there was just one NgoMIV restriction site in the vector. In contrast to that the digestion of P34.3 and P34.4 delivered two bands - whereas the band of the smaller fragments looked like a smier. Because of this obscure and non-satisfying results (besides our assumption that there's something wrong with the marker :) )we decided to sequence the P34.3 vector:

    • c(P34.3): 408.48 ng/µL
    • Volume (plasmid): 5.14 µL
    • Volume (vector): 24.86 µL
    • Name of eppi: HW_34
    • Primer: GATC_std_pTeSp-1

    Picking of Clones from pAAV_iGEM-MCS_mVenus-YFP_Vektor_oben

    • experiment date: 9.6.2010
    • name of investigator: Hanna, Achim
    • plasmid:
      • Vector: name: pAAV_iGEM-MCS_Vektor_oben
      • Insert: name: pOG14_mVenus
    • new vector name: pAAV_iGEM-MCS_mVenus-YFP_Vektor_oben


    The Clones were picked and incubated in 10 mL LB-Medium containing 10µL ampicillin over night at 37°C on a rotary shaker.

    26. Labortag 10.06.2010:

    Site-directed mutagenesis of PstI in ITRs

    1. Site directed mutagenisis



    Mini-Prep of pAAV_iGEM_Venus_YFP and test digestion

    2.1 Mini-Prep of pAAV_iGEM_mVenus_YFP


    Title: pAAV_iGEM_mVenus_
    investigator: Jessy, Achim
    Glycerol Stocks

    Clone 1 Clone 2
    Bacteria strain XL1 blue XL1 blue
    Plasmidname pAAV_iGEM_mVenus pAAV_iGEM_mVenus
    Date 10.06.2010 10.06.2010
    given number B30 B31


    Given Plasmid-Number

    Clone 1 Clone 2
    given number P35 P36


    Title: pAAV_iGEM_mVenus_YFP (guided by Sven)
    investigator: Bea Glycerol Stocks

    Clone 1 Clone 2 Clone 3 Clone 4
    Bacteria strain BL-21 BL-21 BL-21 BL-21
    Plasmidname pAAV_iGEM_mVenus_YFP pAAV_iGEM_mVenus_YFP pAAV_iGEM_mVenus_YFP pAAV_iGEM_mVenus_YFP
    Date 10.06.2010 10.06.2010 10.06.2010 10.06.2010
    given number B32 B33 B34 B35


    Given Plasmid-Number

    Clone 1 Clone 2 Clone 3 Clone 4
    given number P37 P38 P39 P40


    2.2 Test digestion

    • buffer used: 4 ; Restriction-enzymes used: Enzyme XbaI (no. Lab:___) ; Enzyme AgeI (no.Lab:___) ____
    • Plasmid
      • Given Plasmid-Number: P37; DNA concentration: 114,8 ng/µL ;
      • Given Plasmid-Number: P38 ; DNA concentration: 179,8 ng/µL;
      • Given Plasmid-Number: P39 ; DNA concentration: 180,8ng/µL ;
      • Given Plasmid-Number: P40; DNA concentration: 189,2 ng/µL;


    Comments: Clones were picked from trafo-plate in the morning and mini-prep was performed in the late afternoon. Due to this and the fact that bacterial strain was BL-21, DNA-concentrations are quite low.

    Investigators: Bea, Chris W.

    Components Volume/µL Mastermix Sample: P37 Sample: P38 Sample: P39 Sample: P40
    DNA 800 ng - 7,0 µL4,5 µL4,4 µL 4,2 µL
    BSA (10x) yes 1,5 µL 7,5 µL MM 4,5 µLMM 4,5 µLMM 4,5 µL MM 4,5 µL
    Buffer no. 4 (10x) 1.5 µL 7,5 µL """ "
    Enzyme 1 (no. Lab: ) AgeI 0.75 µL 3,75 µL """ "
    Enzyme 2 (no. Lab: ) XbaI 0.5 µL 2,5 µL """ "
    H2</sup>O variable - 3,75 µL0,25 µL6,35 µL 6,55 µL
    Total volume 15 µL - 15 µL15 µL15 µL 15 µL


    • Incubation: 45 min, 37°C


    27. Labortag 11.06.2010:

    in additon to

    Site directed mutagenisis

    on 10.06.10


    transformation
    transformation was performed according to the standard protocol Media:Freiburg10_Cloning Protocol.pdf
    Cells were plated on agar plates (2x 1:100; 2x pellet)containing ampicillin and stored over night at 37°C.

    Sequenzing

    p39 was send to GATC for sequenzing

    • p39: 180,83 ng*µl^-1
    • volume plasmid: 8,3 µl
    • volume water: 21,7 µl
    • primer: GATC_std_pTeSp-1

    28. Labortag 12.06.2010:

    Kira
    The plates were stored in the iGEM shelf @ 4 °C. Even if the transformation was not very successfull, some colonies can be picked and inoculated either tomorrow or on Monday. Note: Site-directed mutagenesis does not generate lots of intact plasmids, so few colonies are normal!! (Tobias)

    29. Labortag 13.06.2010:

    Jessy, Patrick, Hanna
    We weren't able to detect any colonies on the plates - just a few air bubbles :) To be entirely sure, we put them back into the 37°C room over night.

    30. Labortag 14.06.2010:

    pAAV_iGEM-mVenus-YFP:

    The sequencing data of pAAV_iGEM_mVenus-YFP was analyzed. The alignment with the "theoretical" pAAV_iGEM_mVenus-YFP showed that we successfully inserted mVenus-YFP into pAAV_iGEM-MCS. Freiburg10 mVenus-Alignment.jpg

    Site-directed mutagenesis:

    Also today no colonies were detectable on any plates!

    TK-GMK-Plasmid

    Adrian, Hanna
    puB6_V5_His6_clone1 + TK/GMK (P15) will be sequenced again:

    • name: AF 1
    • primer: GATC_std_BGH-reverse
    • volume(plasmid) = 4.25 µL
    • volume (H2</sup>O) = 25.75 µL

    Results (15.06.): Unfortunately just ~ 300 bp were sequenced.

    To do: Therefore more primers have to be designed in order to sequence the whole ~2000 bp TK-GMK fusion construct!


    Zellkultur

    AAV 293 and HT1080 Cells have been splitted and plated out on 10 cm dishes for transfektion. The Cells were accounted by using the Neubauer-Meteringchamber. After harvesting by following the standard protocol the pallet have been resuspendiated in 15ml of DTT medium. 2,5µl of this cell suspension have been mixed with 47.5µl of trypan blue.

    We counted 12,5x 10^6 cells/ml for the T293 AAV cell line and 10x10^6 cells /ml for the HT1080 cell line.

    The AAV 293 cells have been plated out on four dishes with 250µl of the cell suspension, on one plated with 1ml and on another dish with 1.5ml. note that that the date is wrong on the plates and the flasks! Cells have been already plated out on the 14th. of June.

    We also seated two flasks one for each cell line. Therefor we used 1ml of the HT1080 cell suspension and two ml of the the AAV293 cells.

    31. Labortag 15.06.2010:

    Adrian, Achim, Chris W., Hanna

    Solutions for transfection were prepared:

    • 1 x TE-Buffer: 1.2114 g TRIS, 0.2 mL EDTA, 81 mL milipore-H2</sup>O were mixed. pH was adjusted with HCl (1M and 5M) to 7.50. Volume was adjusted to 100 mL with milipore-H2</sup>O. Solution was autoclaved (sterilization by filtration is also possible).
    • 1 M CaCl2: 147.02 g CaCl2 x 2H2</sup>O (M = 147.02 g/mol) was solved in 1 Liter H2</sup>O and sterilized by filtration.
    • 2 x HBS: 0.027 g Na2HPO4, 1.636 g NaCl, 1.19155 g HEPES were dissolved in ~ 30 mL milipore-H2</sup>O. pH was adjusted to 7.10. Volume was adjusted to 100 mL and sterilized by filtration.


    32. Labortag 16.06.2010:

    Sponsoring:

    Anna, Kerstin, Anissa
    Sponsoring letters were examined and modified one more time.

    Sequencing of TK-GMK:

    Hanna
    Two primers were designed and ordered from Sigma-Aldrich. Probably we will receive them on Friday.

    Mega primer PCR:

    Bea, Hanna

    Freiburg10 ITRprimer.jpg

    33. Labortag 17.06.2010:

    Primer designing: VP1 primer for pKEX forward/reverse

    Investigator: Volker
    The construnts that we receieved from PD Dr. Kleinschmidt (DKFZ) are mainly in the pKEX backbone. The sequences of the backbone and the inserts are not availible, for this reason we decided to sequence the constructs. The first step is to sequence from one construct into the backbone. In the second step we will designe primers that bind in the backbone and point into the direction of the insert. These primers will then be used to sequence the inserts of all the constructs.

    Forward primer pKEX in VP1 from bp 4124 to 414

    • VP1 primer for pKEX forward: 5' - CAACAAGTCTGTTAATGTGGAC - 3' 22bp; TM: 50°C; CG% 41%

    Reverse primer pKEX in VP1 from bp 2081 to 2100

    • VP1 primer for pKEX reverse: 5' - GTGGGCCAGGTTTGAGCTTC - 3' 20bp; TM: 54°C CG%: 60%

    The amplicon that should be produced by the primers is 199 bp long.

    Primer designing: CMV_forward/reverse_qPCR

    Investigator: Volker
    Primers for the titering of the CMV region from the literature [Rohr et al., 2002] and [Rohr et al., 2005] were compared and analysed. The sequences from 2002 showed a differce in one nucleotide compared to the other primer and to our sequence. For this reason the second set [Rohr et al., 2005] was ordered.

    • CMV_forward_qPCR: 5' - GGGACTTTCCTACTTGGCA - 3'
    • CMV_reverse_qPCR: 5' - GGCGGAGTTGTTACGACA - 3'
    Obtaining fragments by digestion with AlwNI. Fragments contain left and right ITR respectively

    These primers will be used in a quantitative PCR assay with Sybr-green to measure the genomic titer and the infectious titer of the viral particles we will produce in the future.

    Ordering of required reagents:

    Investigator: Volker

    • Nuclease S1 was ordered from Promega (10000Units for 36,00€)
    • Proteinase K was ordered from Sigma (5mg for 31,30€; BioUltra, ≥30 units/mg protein, lyophilized powder)

    These reagents are required for the procedure to determine the genomic and the infectious titer.

    34. Labortag 18.06.2010: Transfection

    Transfection

    Transfection via calcium phosphat with the prepared solutions (31. Labortag 15.06.2010)

    investigators: Chris W., Hanna, Patrick, Adrian, Volker
    Transfections with the following plasmids:

    1)

    • pAAV_iGEM_mVenus_YFP (glycerol stock B34, P39) P39 has the correct sequence (confirmed)
    • pHelper
    • pAAV_RC

    2)

    • pAAV_iGEM_mVenus_YFP (glycerol stock B33, P38) P38 (we dont know if P39 has the correct sequence!)was used because the amount of P39 was not sufficient enough for four Transfections!
    • pHelper
    • pAAV_RC

    Plasmid concentrations:
    pAAV_RC: 1 µg/µl
    pHelper: 280 ng/µl
    pAAV_iGEM_mVenus_YFP, P39: 180,83 ng/µl
    pAAV_iGEM_mVenus_YFP, P38: 179,75 ng/µl

    Cellculture clone 1 and 2 were transfected with P39. Cellculture clone 3 and 4 were transfected with P38.

    Deviations from the standard protocol (currently incomplete): We could only pipet 3,3 µg (instead of 10 µg) from each of the 3 plasmids into the 15 ml falcons due to insufficient amount of plasmid.

    Adrian tried to examine the precipation of the CaCl2+ DNA Clusters. We have to optimize the pH of our 2xHBS at the moment it is 11.12

    Theoretical: Digestion of pAAV_MCS for PCR to design ITR BioBrick

    investigator: Bea
    idea:

    • Digest vector in order to obtain two fragments which contain the left and the right ITR respectively.
    • Separation (Agarose-gel) and gel extraction of fragments
    • Perform PCR with iGEM-Primers (forward primer contains EcoRI and Xbai; reverse primer contains SpeI and PstI)
    • to do:
      • design primers
      • digestion of pAAV_MCS etc.

    Theoretical: Digestion of pAAV_MCS with AlwNI produces two fragments which can be separated by agaorse gel.

    Obtaining fragments by digestion with AlwNI. Fragments contain left and right ITR respectively