Team:Heidelberg/Notebook/Killing I/Notebook/week10

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   Home    Team   Overview    Advisors  </a></li>  Undergraduates  </a></li>  University  </a></li>  DKFZ  </a></li>  BioQuant  </a></li>  BioRegion Rhein-Neckar  </a></li> </ul> </li>  Project</a> <ul class="DropDownMenu" id="MB1-DDM1">  Overall Project  </a></li>  Material & Methods  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Sensing"> Sensing  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Killing_I"> Killing I  - Phages  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Killing_II"> Killing II - Colicin  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Visualization"> Visualization  </a></li> </ul> </li>

<li> <a href="http://2008.igem.org/Team:Heidelberg/Parts" style="color: white">Parts</a> <ul class="DropDownMenu" id="MB1-DDM2"> <li><a href="http://2008.igem.org/Team:Heidelberg/Parts"> Submitted Parts  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Parts/Characterization"> Characterization  </a></li> </ul> </li> <li> <a href="http://2008.igem.org/Team:Heidelberg/Modeling" style="color: white">Modeling</a> <ul class="DropDownMenu" id="MB1-DDM3"> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling"> Overview  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling/Chemotaxis"> Chemotaxis-Colicin  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling/Phage"> Phage Dynamics model  </a></li> </ul> </li> <li> <a href="http://2008.igem.org/Team:Heidelberg/Notebook/Overview" style="color: white">Notebook</a> <ul class="DropDownMenu" id="MB1-DDM5"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"> Sensing >  </a> <ul class="SideMenu" id="MB1-DDM2-SM1"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"> Killing I - Phages >  </a> <ul class="SideMenu" id="MB1-DDM2-SM2"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"> Killing II - Colicin >  </a> <ul class="SideMenu" id="MB1-DDM2-SM3"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/visualization"> Visualization  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/material"> Material & Methods  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/team_meetings"> Team Meetings  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/seminar"> Seminar on Synthetic Biology  </a> </ul> </li> <li style="width: 160px"> <a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview" style="color: white">Human Practice</a> <ul class="DropDownMenu" id="MB1-DDM4"> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview"> Project Overview  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Phips_the_Phage"> Phips the Phage  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Essay"> Essay  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Surveys"> Surveys  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Open_Day"> Open Day  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Nobel_Prize"> Nobel Prize  </a></li> </ul> </li>

<li> <a href="http://2008.igem.org/Team:Heidelberg/Sponsors" style="color: white">Sponsors</a> </li> </ul>

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Week 10

Proceeding of the overnight ligations

 * 8 transformation of the CmR overnight ligations
 * 3 control transformation of Term1, CmR3 and pBluesript backbone

Proceeding of the minipreps from friday

 * digestions of CmR in pBlue Miniprep to check for sequencing
 * digestion with SacI, KpnI and EcoRI
 * expected bands: 2859, 586, 272


 * digestion with DraI
 * expected bands: 1480, 1187, 692, 339, 19


 * Gel
 * top:
 * lane1-3: CmR1 (undigested, SacI/KpnI/EcoRI, DraI)
 * lane4-6: CmR2
 * lane7-10: CmR3a
 * lane11-13: CmR3b
 * bottom
 * lane1-3: CmR4 (undigested, SacI/KpnI/EcoRI, DraI)
 * lane4-6: CmR5
 * lane7-10: CmR7
 * lane11-13: CmR8


 * according to the digestions we have the chloramphenicol resistance cassette in pBluescript


 * Retrafo of CmR minipreps because we don't have enough DNA and unfortunately no glycerol stocks
 * 3µl of CmR 1, 2, 3a, 3b

Cloning of CmR in standard plasmid

 * PCR of CmR with CmR_prefix_fw and CmR_suffix_fw

25µl Phusion Master Mix 2x 1µl CmR_suffix_fw 1µl CmR_prefix_rev 1µl Maxiprep pBlue with insert (stock: ~200ng/µl, dilution: 1:20-->10ng) 22µl water - 50µl

PCR protocol 98°C 1min 98°C 10s | 61°C 10s | 26x 72°C 45s | 72°C 5min 4°C for ever


 * Gel


 * expected size: 851bp+43bp=894bp
 * lane0:ladder
 * lane1: CmR1
 * lane2: CmR2
 * lane3: CmR3a
 * lane4: CmR3b
 * lane5: CmR4
 * lane6: CmR5
 * lane7: CmR7
 * lane8: CmR8


 * cuted out CmR band and gel extraction kit

Cloning of cI in standard plasmid

 * unwanted EcoRI restriction site in cI from geneart was mutated using standard protocol


 * lane 1+2: mutated cI digested with EcoRI --> mutagenesis was succesfull

--> digested cI and pSB1A2 backbone was cut out and ligated (15 min, RT) --> transformation in E.coli Top10 --> inoculation of liquid cultures --> miniprep --> send for sequencing and controll digest with EcoRI/PstI results: sequencing and digestion pattern is correct
 * maxiprep of this cI as well as J01003 in pSB1A2 were digested with EcoRI/PstI and XbaI/PstI to ligate this mutated cI in a standard plasmid (pSB1A2)
 * lane 1-4: cI digested with EcoRI/PstI
 * lane 5-9: cI digested with XbaI/PstI (lane 7 is ladder)
 * lane 10+11: J01003 digested with XbaI/PstI
 * lane 12+13: J01003 digested with EcoRI/PstI
 * seqeuncing was succesfull
 * digestion with EcoRI/PstI
 * lane 1-6: mutated cI in pSB1A2 digested with EcoRI/PstI

Characterization of oriT

 * Inoculate the cells for conjugation test
 * 5ml LB/chloramphenicol + glycerol stock Top10 pBAD 33
 * 5ml LB/kanamycin/ampicilin + 1 colony Cotransformation Top10 J01103+pUB307(12)
 * 5ml LB/kanamycin/ampicilin + 1 colony Cotransformation Top10 J01103+pUB307(22)
 * 5ml LB/kanamycin/ampicilin + 1 colony Cotransformation Top10 J01103+pUB307(13)
 * 5ml LB/kanamycin/ampicilin + 1 colony Cotransformation Top10 J01103+pUB307(23)

Proceeding of cloning CmR in pBluescript

 * a lot of single colonies of the transformation of the new ligations
 * a lot of colonies on the retrafo
 * inoculation of liquid culture of the 4 retrafos and the first 4 new ligations
 * agar plates with new ligations are stored at 4 °C

Cloning of CmR in standard plasmid

 * Digestion of CmR pcr product with PstI, XbaI

5µl NEB3 5µl BSA 10µl PCR product CmR (app. 50ng/µl (from gel) ) 1 µl PstI 1,5µl XbaI 32,5µl water


 * 4 samples: 1,2, 3a, 3b


 * 2h at 37°C
 * PCR purification kit


 * ligation of digested CmR pcr product in pSB1A2, 30min at room temperature
 * transformation in TOP10, plated out on CmR plates
 * ligation was done overnight as well

Phage cloning strategy two

 * Digestion of KpnI mutagenesis PCR

3µl DNA 5µl NEB1 5µl BSA 1µl KpnI 1µl AgeI 36µl water


 * 4 digestions with following mutagenesis pcr samples: 1,3,4,6


 * Gel


 * mutagensis pcr successful: 1690bp, 5050bp
 * -->cut out 5050bp band
 * mutagensis pcr unsuccessful: 1684bp, 1690bp, 3366bp


 * lane0: DNA ladder mix
 * lane1-3: digested pcr sample 1
 * lane4-6: digested pcr sample 3
 * lane7-9: digested pcr sample 4
 * lane10-12: digested pcr sample 6


 * Digestion of CmR out of CmR in pBluescript with KpnI SacI

5µl NEB1 5µl BSA 3µl CmR miniprep 1,5µl KpnI 1,5µl SacI 34µl water

--> digestion of CmR miniprep 1,2,4,5


 * Gel
 * expected: 858bp, 2859bp
 * -->cut out 858bp band


 * lane0:ladder
 * lane1-3: digested miniprep 1
 * lane4-6: digested miniprep 2
 * lane7-9: digested miniprep 4
 * lane10-12: digested miniprep 5


 * ligation of CmR (KpnI/SacI), GFP (SacI/AgeI), pBluescript (KpnI/AgeI) 30min at room temperature
 * transformation in TOP10
 * ligation was done at 16°C over night as well

Characterization of oriT
Donor: overnight culture Cotransformation Top10 oriT+pUB307(12), (22), (13), (23) Recipient: overnight culture Top10 pBAD 33 100ul overnight culture Top10 oriT+pUB307(12) 100ul overnight culture Top10 oriT+pUB307(22) 100ul overnight culture Top10 oriT+pUB307(13) 100ul overnight culture Top10 oriT+pUB307(23) 100ul overnight culture Top10 oriT+pUB307(12) 100ul overnight culture Top10 oriT+pUB307(22) 100ul overnight culture Top10 oriT+pUB307(13) 100ul overnight culture Top10 oriT+pUB307(23) 100ul overnight culture Top10 pBAD 33 100ul conjugation mix (12) 100ul conjugation mix (13) 100ul conjugation mix (22) 100ul conjugation mix (23) All LB/Cm positive; all LB/Kan+Amp positive; LB/Amp+Cm with donor or recipient negative; LB/Amp+Cm with conjugation mix positive -> like expectation
 * Qualitatively test for oriT
 * Centrifuge 500ul overnight culture in 1.5ml eppi for 2min at 13000rpm
 * Wash the pellet twice with LB medium
 * Resolve the pellet in 500ul LB medium
 * Mix 500ul washed recipient cell suspension with 500ul washed donor cell suspension in 2ml eppi
 * Vortex
 * Incubate the mix at 37°C for 1hr
 * Plates:
 * LB/Cm: 100ul overnight culture Top10 pBAD 33
 * LB/Kan+Amp:
 * LB/Amp+Cm:
 * Result:


 * Inoculate cells for conjugation test
 * 5ml LB/chloramphenicol + 10ul overnight culture Top10 pBAD 33
 * 5ml LB/kanamycin/ampicilin + 10ul overnight culture Cotransformation Top10 oriT+pUB307(12)
 * 5ml LB/kanamycin/ampicilin + 10ul overnight culture Cotransformation Top10 oriT+pUB307(22)
 * 5ml LB/kanamycin/ampicilin + 10ul overnight culture Cotransformation Top10 oriT+pUB307(13)
 * 5ml LB/kanamycin/ampicilin + 10ul overnight culture Cotransformation Top10 oriT+pUB307(23)

Phage cloning strategy two

 * transformation of overnight ligations in TOP10


 * screening pcr to check if ligation was successful using GFP_new_fw and GFP_new_rv
 * pcr was not successful, gel included no pcr product

Characterize of oriT
Donor: overnight culture Cotransformation Top10 J01103+pUB307(12) OD(600nm): 2.844 Recipient: overnight culture Top10 pBAD 33 OD(600nm): 3.346 100ul donor overnight culture 100ul recipient overnight culture
 * Quantitatively test for oriT
 * Centrifuge 250ul overnight culture in 1.5ml eppi for 2min at 13000rpm, 10samples donor, 10samples recipient
 * Wash the pellet twice with LB medium
 * Resolve the pellet in 250ul LB medium
 * Centrifuge the washed recipient for 2min at 13000rpm, discard the fluid
 * Add the washed donor suspension
 * Vortex and resolve the pellet
 * Centrifuge the mix for 1min at 13000rpm
 * Resolve the pellet in 100ul LB
 * Put membrane filter on the LB agar
 * Pipett the suspension on membrane filter (10samples)
 * Incubate the plates with membrane filter at 37°C
 * Put directly one membrane filter into 1ml LB in an 1.5ml eppi
 * Vortex the eppi for 30sec, dilute for 10-4 and 10-5, plate them out on LB/Amp+Cm plates (0min)
 * After 6, 12, 18, 24, 30, 36, 42, 48, 54min repeat the last two steps.
 * Negative control plates:
 * LB/Cm+Amp:
 * Cell number determination
 * LB/Cm: 100ul 10-6 recipient overnight culture
 * LB/Kan+Amp: 100ul 10-6 donor overnight culture
 * Result:
 * Negative control: negative
 * Colony on LB/Cm: 324 (Titer of recipient: 3.24e9/ml)
 * Colony on LB/Kan+Amp: 373 (Titer of donor: 3.73e9/ml)
 * Colony on other LB/Cm+Amp plates:
 * 10-4 dilute: impossible for counting
 * 10-5 dilute:
 * 36min to 54 min: impossible for counting
 * Make glycerol stock for Cotransformation Top10 oriT+pUB307
 * 1ml overnight culture of Top10 oriT+pUB307 (12)+ 150ul 80% glycerol
 * Vortex
 * 1hr RT
 * Freeze at -80°C

Cloning of CmR in standard plasmid

 * miniprep of 12 from the 20 overnight cultures (CmR in pSB1A2)
 * sample nr. 1.1-1.3, 2.1-2.3, 3.1-3.3, 4.1-4.3

4µl EcoRI buffer 4µl BSA 1µl EcoRI 1µl PstI 3µl Miniprep DNA 27µl water
 * digestion of the 12 minipreps with EcoRI and PstI


 * Gel
 * expected fragments: 292,601,2038bp


 * lane0: DNA ladder mix
 * lane1: 1.1
 * lane2: 1.2
 * lane3: 1.3
 * lane4: 2.1
 * lane5: 2.2
 * lane6: 2.3
 * lane7: 3.1
 * lane8: 3.2
 * lane9: 3.3
 * lane10: 4.1
 * lane11: 4.2
 * lane12: 4.3


 * mutagenesis pcr with 2.2 and 2.3 overnight to remove EcoRI restriction site

5µl Pfu buffer 2µl CmR_EcoRI_mut_fw 2µl CmR_EcoRI_mut_rv 1,5µl dNTPs 1µl Miniprep, diluted 1:10 1µl Pfu (not turbo!) 37.5µl water

PCR protocol 95°C 30s 95°C 30s   | 55°C 45s   | 16x 68°C 6.5min | 4°C for ever

Phage cloning strategy two

 * screening pcr with CmR_prefix_fw and CmR_suffix_rv primer (using Taq)
 * Gel
 * lane0: dna ladder
 * lane1: colony 1+2
 * lane2: colony 3+4
 * lane3: colony 5+6
 * lane4: colony 7+8
 * lane5: colony 9+10
 * lane6: colony 11+12
 * lane7: colony 13+14


 * expected sizes:
 * pBlue with insert: 1668bp
 * CmR cassette only: 852bp
 * GFP cassette only: 919bp


 * colonys 1,2,3,4,9,10 look good

Cloning of CmR in standard plasmid

 * proceeding of EcoRI mutagenesis pcr
 * 3h DpnI digestion at 37°C
 * transformation in TOP10

--> miniprep 1.1, 2.1, 2.2 look good
 * digestion of the 12 minipreps (CmR in pSB1A2) with PstI/XbaI
 * expected fragments: 2053bp, 878bp
 * Gel
 * lane0: ladder
 * lane1-12: miniprep 1.1 - 4.3

Phage cloning strategy two

 * Screening PCR wirh oriT_fw and CmR_suffix_rv (using Taq)
 * expected sized:
 * pBlue/insert: 2150bp
 * pBlue/GFP/CmR: ca. 1.3kb




 * Gel
 * lane0: DNA ladder mix
 * lane1-12: sample 1-12


 * lane 7 looks good ~1.3kb

Cloning of oriT in standard plasmid

 * Miniprep of oriT (from RP4)
 * PCR with oriT prefix/suffix primer using Phusion
 * expected size: ~500bp
 * 4 pcr samples


 * lane0: dna ladder mix
 * lane1-4: oriT pcr probe 1-4


 * PCR purification kit
 * Digestion with PstI/EcoRI
 * PCR purification kit
 * Ligation in pSB1A2 20min at RT (after 40min heat inactivation at 65°C)
 * Transformation
 * transformation was done on Cm plates although there is no Cm resistance (failure!)
 * -->make transformation again on sunday and plate out on Amp plates

Proceeding of cloning CmR in standard plasmid

 * inoculation of Mutagensis PCR 2.2 (Cmr Std) (only one colony was grown)
 * Mutagenesis PCR of 1.1, 1.2 and 2.2 (CmR Std) using Turbo Pfu

5µl Pfu buffer 2µl CmR_EcoRI_mut_fw 2µl CmR_EcoRI_mut_rv 1,5µl dNTPs 1µl Miniprep, diluted 1:10 1µl turbo Pfu 37.5µl water

PCR protocol 95°C 30s 95°C 30s   | 55°C 45s   | 16x 68°C 7min | 4°C for ever

Phage cloning strategy two

 * inoculation of pBluescript+GFP+CmR 1-6,9,10 (nach fraktioniertem Ausstrich)
 * redo Screening PCR wirh oriT_fw and CmR_suffix_rv (using Taq)
 * no bands visible! (only primers)

Proceeding of cloning CmR in standard plasmid

 * Miniprep of Mutagenesis PCR 2.2
 * Digestion of Mutagenesis PCR Miniprep 2.2 with PstI, EcoRI
 * -->digestion showed that the mutagenesis PCR did not work


 * DpnI digestion of mutagenesesis PCR (2h at 37°C)
 * transformation of mutagenesis PCR in TOP10

Proceeding of cloning oriT in standard plasmid

 * redo transformation and plate out on Amp plates

Phage cloning strategy two

 * Miniprep of pBlue+GFP+CmR 1-6,9,10