1283 pDB.His.MBP.Ent
Cloning of 1283 pDB.His.MBP.Ent
[edit]Purpose: Cloning of 1283 pDB.His.MBP.Ent
Date: July 31, 2012
Notebook:
Page:
Clone ID: 1283
Experimenters: Jennifer Truong
Results
[edit]Cloning
[edit]Date: July 31, 2012
PCR MBP from pMAL-p5E using
1 ul of 10 uM MBP-Xba1-F1 cgatcgTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAatgggcagcagccatc
1 ul of 1 uM MBP-F2 GATATAatgggcagcagccatcatcatcatcatcacATGAAAATCGAAGAAGGTAAAC
1 ul of 10 uM MBP-Nde1-R gtgctatCATATGGCCCATGGACGGTACCTTGTCATCGTCATCCCCGAGGTTGTTGTTATTGTTATTGTTG
Digest PCR product with Xba1 + Nde1, ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14
See lane 2, expected size ~1400 bp
Cut out band and digested
Digest PCR product with Xba1 + Nde1 (lane 1), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 2)
Cut out bands, purify and ligate together overnight
Date: August 7, 2012
Sequencing came back contaminated, started all over
See lanes 1 and 2
PCR MBP from pMAL-p5E using
1 ul of 10 uM MBP-Xba1-F1 cgatcgTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATAatgggcagcagccatc
1 ul of 1 uM MBP-F2 GATATAatgggcagcagccatcatcatcatcatcacATGAAAATCGAAGAAGGTAAAC
1 ul of 10 uM MBP-Nde1-R gtgctatCATATGGCCCATGGACGGTACCTTGTCATCGTCATCCCCGAGGTTGTTGTTATTGTTATTGTTG
Digest PCR product with Xba1 + Nde1, ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14
See lane 2, expected size ~1400 bp
Cut out band and digested
Digest PCR product with Xba1 + Nde1 (lane 1), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 2)
Cut out bands, purify and ligate together overnight
Date: August 9, 2012
Digestion was messed up from earlier because of the gel, started all over
Cut out lanes 1 and 2 for digestion, save 3 and 4 in box just incase something happens
Date: August 17, 2012
Digest PCR product with Xba1 + Nde1 (lane 7), ligate to Xba1 + Nde1 digested vector pDB.His.BMP, V14 (lane 8)
Cut out bands, purify and ligate together overnight
Colony Screening
[edit]Date: August 3, 2012
Digested MiniPrep plasmids with RE Xba1 + Nde1 in lanes 1 - 6 (samples 1-6 respectively)
Digested sample 1 with Xba1 + Nco1 in lane 8, and digested sample 1 with Xba1 + Kpn1 in lane 9
All correct sizes, send sample 1 to sequencing
Clones
These are contaminated clones Date: 2012
Clones 2, 3, 4, 5 and 6 may be correct
Result intepretation/Summary
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Conclusion
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