Team:Harvard/Template:NotebookDataJuly2

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None of the colonies displayed any bands, except for primer residues. Lane 16 contained the selection plasmid, on which we performed [[#Gel purification of PCR product|a successful PCR]] on Friday, so we definitely expected a band at 709 bp. Because the positive controls did not have a band at 709 bp, we suspect that something went wrong with the PCR. We plan on re-running the PCR tomorrow.</div>
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None of the colonies displayed any bands, except for primer residues. Lane 16 contained the selection plasmid, on which we performed [https://2011.igem.org/Team:Harvard/Protocols#PCR_purification a successful PCR] on Friday, so we definitely expected a band at 709 bp. Because the positive controls did not have a band at 709 bp, we suspect that something went wrong with the PCR. We plan on re-running the PCR tomorrow.</div>
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===Team Web===
===Team Web===
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*Design [https://spreadsheets.google.com/spreadsheet/ccc?key=0ApTl36bX3P7qdFo3SEFXMkNCZ3ZBX1hBQUZqWUpiV2c&pli=1#gid=0 Google Doc]</div>
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*Looked at good wikis from last year. </div>
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==July 12th==
==July 12th==
===Team ZF===
===Team ZF===
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Even after the gel purification, our bands were streaky. We decided to redo the PCR, placing all the reactions in single tubes, performing a touchdown PCR instead. Our annealing temperature started at 70*, repeated for 2 cycles, and then decreased 2*, every two cycles, until it reached 58* (the optimal annealing temperature), where it remained until the 25 cycles were complete. We left this PCR going overnight. (See [[#Results of ultramer touchdown PCR|July 13th]] for the gel image.)
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Even after the gel purification, our bands were streaky. We decided to redo the PCR, placing all the reactions in single tubes, performing a touchdown PCR instead. Our annealing temperature started at 70*, repeated for 2 cycles, and then decreased 2*, every two cycles, until it reached 58* (the optimal annealing temperature), where it remained until the 25 cycles were complete. We left this PCR going overnight. (See July 13th for the gel image.)
====PCR of expression plasmid cross-junction====
====PCR of expression plasmid cross-junction====
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We also performed a second PCR on colony nine (the colony we decided with which we decided to proceed) to confirm that our omega+zif268 in the spec backbone was successfully inserted into the ''E. coli''. The expected product was the cross-junction on the plasmid, approximately 1.4 kb. We used the same recipe as that used on [[#PCR of omega+zif268 with overhangs|July 9th]], except with a forward primer of PZE23G-3581 F and a reverse primer of PZE23G-2133 R, and had a 55* annealing temperature and an extension time of 45 seconds.  
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We also performed a second PCR on colony nine (the colony we decided with which we decided to proceed) to confirm that our omega+zif268 in the spec backbone was successfully inserted into the ''E. coli''. The expected product was the cross-junction on the plasmid, approximately 1.4 kb. We used the same recipe as that used on July 9th, except with a forward primer of PZE23G-3581 F and a reverse primer of PZE23G-2133 R, and had a 55* annealing temperature and an extension time of 45 seconds.  
After running an e-gel, we confirmed that our w+zif268 was inserted into the plasmid.
After running an e-gel, we confirmed that our w+zif268 was inserted into the plasmid.

Latest revision as of 18:48, 3 August 2011