Team:Freiburg Bioware/testpage

• Materials

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

Given Plasmid-Number

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 H_{2</sup>O (Volume on obtained sheet)were added.}
  • expression part MCS_for (charge-no: ST00114066): 165 µL Millipore H_{2</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 H_{2</sup>O (final volume usually 100 µl).}
• Mix together(into PCR-tube):

• 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 :

• 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

• Expected size of fragments

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Gelextraction:

Gel measurement:

• 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:

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

Given Plasmid-Number

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:

• 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

• Marker: GeneRuler ladder mix

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

Given Plasmid-Number

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 dH_2</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):

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:

• Vector: 4,16 µl
• Insert: 4,84 µl

• Vector: 6 µl
• Insert: 3 µl

continuation of site-directed mutagenesis

Digestion with DpnI:

Trafo:

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!

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

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

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;

• 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

• Marker: GeneRuler ladder mix

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.