Team:Heidelberg/Notebook/Killing I/Notebook/week11

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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>

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Week 11

Proceeding of phage cloning strategy two

 * Digestion of the miniprep 1-6,9,10 from sunday and the original pBluescript with insert


 * Digestion with XbaI/XhoI (top)
 * normal: 4549, 2157, 34
 * new: 3945, 2898


 * Digestion with SacI/SpeI (bottom)
 * normal: 4549, 2157, 34
 * new: 4549, 1331, 929, 34


 * Gel
 * lane0: dna ladder mix
 * lane1: sample 1
 * lane2: sample 2
 * lane3: sample 3
 * lane4: sample 4
 * lane5: sample 5
 * lane6: sample 6
 * lane9: sample 9
 * lane10: sample 10
 * lane11: pBluescript with insert


 * PCR with CmR_suffix and CmR_prefix (top)
 * normal: 1668bp
 * new: 852bp, 919bp


 * PCR with oriT_prefix and CmR_suffix (bottom)
 * normal: 2150bp
 * new: 1329bp


 * Gel
 * lane0: dna ladder mix
 * lane1: sample 1
 * lane2: sample 2
 * lane3: sample 3
 * lane4: sample 4
 * lane5: sample 5
 * lane6: sample 6
 * lane9: sample 9
 * lane10: sample 10
 * lane11: pBluescript with insert


 * --> we do not have a working GFP/CmR in pBlue/insert!!! --> do the ligation again (beginng from the mutagenesis pcr)

Proceeding of cloning CmR and oriT in standard plasmid

 * inoculation of CmR Std. Mutagenesis PCR sample and oriT Std. colonies

Proceeding of cloning CmR and oriT in standard plasmid

 * Miniprep of 6 oriT and 5 CmR Std. samples
 * digestion with EcoRI/PstI (to cut out insert of pSB1A2)
 * lane0: dna ladder mix
 * lane1-6: oriT 1-6
 * lane7: 1kb dna ladder plus
 * lane8: CmR Std. Mut 1.1.1
 * lane9: CmR Std. Mut 1.1.2
 * lane10: CmR Std. Mut 1.2.2
 * lane11: CmR Std. Mut 2.2.1
 * lane12: CmR Std. Mut 2.2.2


 * expected fragments:
 * oriT: 2000bp, 500bp
 * CmR: 2000bp, ca. 900bp


 * -->no oriT is right


 * -->CmR 1.1.1, 1.1.2, 2.2.1, 2.2.2 look good
 * -->sequencing of 2.2.1 and 2.2.2
 * sequencing results match perfect


 * Three ligations of oriT pcr product with pSB1A2 backbone (PstI, XbaI) (1:2.5, 2:5, 3:7.5 (µl, vector:insert))
 * 40min at RT
 * transformation, plated out on Amp plates


 * Screening PCR of oriT agar plates
 * pcr with standard plasmid primers (VF2, VR) using Taq
 * 12 pcr samples (1-6 only one colony, 7-12 three colonies)
 * Gel
 * lane0: dna ladder mix
 * lane1-12: screening sample 1-12
 * expected fragment length: ca. 500-600bp
 * -->sample 3 and 5 look good
 * -->inoculation of overnight cultures
 * -->sequencing of 3 and 5
 * sequencing results match perfect

Phage cloning strategy two

 * Mutagenesis PCR of pBlue with insert to remove KpnI restriction site
 * using turbo Pfu
 * elongation: 12,5min
 * DpnI digestion 2h at 37°C
 * transformation in TOP10, plated out on Cm plate

Phage cloning strategy one

 * mutation of old insert in pBluescript
 * pBluescript + insert was cut with BamHI --> the resulting backbone cut out of the gel and religated. With this vector 2 mutagenesis PCRs were done to eleminate the two remaining XbaI restriction sites.
 * The resulting vector was digested with XbaI/XhoI to get the insert with the correct restriction sites for ligation into lambda phage

Characterization of oriT
5ml LB/chloramphenicol + glycerol stock Top10 pBAD 33 5ml LB/kanamycin/ampicilin +glycerol stock Top10 J01103+pUB307
 * Inoculate the cells for conjugation test

Phage cloning strategy two

 * inoculation of KpnI mutagenesis PCR samples --> Miniprep
 * Digestion with KpnI/AgeI
 * Gel
 * Gel purification kit

Characterization of oriT
Donor: overnight culture Top10 oriT+pUB307 OD(600nm): 2.08 Recipient: overnight culture Top10 pBAD 33 OD(600nm): 2.24 100ul donor overnight culture 100ul recipient overnight culture 5ml LB/chloramphenicol + glycerol stock Top10 pBAD 33 5ml LB/chloramphenicol + 1colony MG1655 pBAD 33 5ml LB/chloramphenicol + 1 colony stock DH5alpha pBAD 33 15ml LB/kanamycin/ampicilin +glycerol stock Top10 oriT+pUB307
 * Quantitatively test for oriT
 * Centrifuge 350ul overnight culture in 1.5ml eppi for 2min at 13000rpm, 8samples donor, 8samples recipient
 * Wash the pellet twice with LB medium
 * Resolve the pellet in 350ul 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 (8samples)
 * 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 10-5 and 10-6, plate them out on LB/Amp+Cm plates (0min)
 * After 6, 12, 18, 24, 30, 36, 42 min 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: 238 (Titer of recipient: 2.38e9/ml)
 * Colony on LB/Kan+Amp: 99 (Titer of donor: 0.99e9/ml)
 * Colony on other LB/Cm+Amp plates:
 * 10-5 dilute:
 * 10-6 dilute:
 * Inoculate the cells for conjugation test

Phage cloning strategy two

 * overnight ligation of pBluescript/insert backbone, GFP and CmR

Characterization of oriT
Donor: overnight culture Top10 oriT+pUB307 OD(600nm): 1.772 Recipient: overnight culture Top10 pBAD 33 OD(600nm): 2.472 overnight culture MG1655 pBAD 33 OD(600nm): 4.412 overnight culture DH5alpha pBAD 33 OD(600nm): 3.876 100ul donor overnight culture 100ul recipient overnight culture ( x 3) Top10:179 (Titer of recipient: 1.79e9/ml) MG1655:242 (Titer of recipient: 2.42e9/ml) DH5alpha:196 (Titer of recipient: 1.96e9/ml)
 * Quantitatively test for oriT
 * Centrifuge overnight culture in 1.5ml eppi for 2min at 13000rpm, 24 samples donor, 400ul/sample; 8samples Top10 recipient, 340ul/sample; 8samples MG1655 recipient, 230ul/sample; 8samples DH5alpha recipient, 260ul/sample;
 * Wash the pellet twice with LB medium
 * Resolve the pellet in 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 (8samples x 3tests)
 * 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 10-5 and 10-6, plate them out on LB/Amp+Cm plates (0min)
 * After 6, 12, 18, 24, 30, 36, 42 min repeat the last two steps.
 * Negative control plates:
 * LB/Cm+Amp:
 * Cell number determination
 * LB/Cm: 100ul 10-6 recipient overnight culture ( x 3)
 * LB/Kan+Amp: 100ul 10-6 donor overnight culture<b>
 * Result:
 * Negative control: negative
 * Colony on LB/Cm:
 * Colony on LB/Kan+Amp: 136 (Titer of donor: 1.36e9/ml)
 * Colony on other LB/Cm+Amp plates:
 * 10-5 dilute:

'*':10-6 dilute

Phage cloning strategy two

 * transformation of overnight ligations in TOP10

Phage cloning strategy two

 * inoculation of colonies from the transformation

Phage cloning strategy two

 * Miniprep

Characterization of oriT
5ml LB/chloramphenicol + glycerol stock Top10 pBAD 33 5ml LB/chloramphenicol + 1colony MG1655 pBAD 33 5ml LB/chloramphenicol + 1 colony stock DH5alpha pBAD 33 15ml LB/kanamycin/ampicilin +glycerol stock Top10 oriT+pUB307
 * Inoculate the cells for conjugation test