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Team:Heidelberg/Notebook/Killing I/Notebook/week11
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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
- 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
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)
- 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
- expected fragments:
- -->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
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
old phage cloning strategy
- 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
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
