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

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Week 11

Contents

Monday, 10/13/08

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

Hd-phage-08-10-13-digestion-pBlueGFPCmR.jpg

    • 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

Hd-phage-08-10-13-pcr-pBlueGFPCmR.jpg

    • 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





Tuesday, 10/14/08

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)

Hd-phage-08-10-14-digestion-oriT-CmR.jpg

    • 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

Hd-phage-08-10-14-screening-pcr-oriT.jpg

    • 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

Hd-phage-08-10-17 pBlue insert fully mutated XbaI-XhoI.jpg

Characterization of oriT

  • Inoculate the cells for conjugation test

5ml LB/chloramphenicol + glycerol stock Top10 pBAD 33
5ml LB/kanamycin/ampicilin +glycerol stock Top10 J01103+pUB307

Wednesday, 10/15/08

Phage cloning strategy two

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

Characterization of oriT

  • Quantitatively test for oriT

Donor: overnight culture Top10 oriT+pUB307 OD(600nm): 2.08
Recipient: overnight culture Top10 pBAD 33 OD(600nm): 2.24

    • 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:


100ul donor overnight culture
100ul recipient overnight culture

    • 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:
Time Colony
0 9
6 20
12 94
18 172
24 262


      • 10-6 dilute:
Time Colony
30 145
36 179
42 217


  • Inoculate the cells for conjugation test

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

Thursday, 10/16/08

Phage cloning strategy two

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

Characterization of oriT

  • Quantitatively test for 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

    • 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:

100ul donor overnight culture
100ul recipient overnight culture ( x 3)

    • Cell number determination
      • LB/Cm: 100ul 10-6 recipient overnight culture ( x 3)
      • LB/Kan+Amp: 100ul 10-6 donor overnight culture


  • Result:
    • Negative control: negative
    • Colony on LB/Cm:

Top10:179 (Titer of recipient: 1.79e9/ml)
MG1655:242 (Titer of recipient: 2.42e9/ml)
DH5alpha:196 (Titer of recipient: 1.96e9/ml)

    • Colony on LB/Kan+Amp: 136 (Titer of donor: 1.36e9/ml)
    • Colony on other LB/Cm+Amp plates:
      • 10-5 dilute:
Time Top10 MG1655 DH5alpha
0 9 0 1
6 10 0 1
12 19 0 0
18 25 2 3
24 112 0 100
30 387 8 410
36 740 72 1340*
42 223 1640*


'*':10-6 dilute

Friday, 10/17/08

Phage cloning strategy two

  • transformation of overnight ligations in TOP10


Saturday, 10/18/08

Phage cloning strategy two

  • inoculation of colonies from the transformation


Sunday, 10/19/08

Phage cloning strategy two

  • Miniprep

Characterization of oriT

  • Inoculate the cells for conjugation test

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


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