Team:Freiburg Bioware/testpage

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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.