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

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===old phage cloning strategy===
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===phage cloning strategy one===
* mutation of old insert in pBluescript
* 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.
* 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
* The resulting vector was digested with XbaI/XhoI to get the insert with the correct restriction sites for ligation into lambda phage
[[Image:Hd-phage-08-10-17_pBlue_insert_fully_mutated_XbaI-XhoI.jpg]]
[[Image:Hd-phage-08-10-17_pBlue_insert_fully_mutated_XbaI-XhoI.jpg]]
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==Wednesday, 10/15/08==
==Wednesday, 10/15/08==

Revision as of 11:39, 29 October 2008

<< Week 10 Overview Week 12 >>


Week 11

Contents

Monday, 10/13/08

Proceeding of new phage cloning strategy

  • 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




new phage cloning strategy

  • 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

Wednesday, 10/15/08

new phage cloning strategy

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


Thursday, 10/16/08

new phage cloning strategy

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


Friday, 10/17/08

new phage cloning strategy

  • transformation of overnight ligations in TOP10


Saturday, 10/18/08

new phage cloning strategy

  • inoculation of colonies from the transformation


Sunday, 10/19/08

new phage cloning strategy

  • Miniprep


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