Team:Harvard/Template:NotebookDataJuly2

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*The concentration of the w+zif268 was 54.3 ng/ul.
*The concentration of the w+zif268 was 54.3 ng/ul.
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Using [[Protocols#Isothermal_assembly|our protocols]] we determined we needed:
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Using [https://2011.igem.org/Team:Harvard/Protocols#Isothermal_assembly our isothermal assembly protocol] we determined we needed:
*0.63 ul backbone (length: ~2.2 kb)
*0.63 ul backbone (length: ~2.2 kb)
*0.59 ul w+zif268 (length: ~0.7 kb)
*0.59 ul w+zif268 (length: ~0.7 kb)
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*500 ul LB
*500 ul LB
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We plated the cells, [[Protocols#Cultures|using this protocol]], on spec plates, one of which contained 50 ul of cells and the other 150 ul of cells. The cells have been incubated at 37*, and will be left there overnight. Cells that have taken up the plasmid have spec-resistance and should be able to survive. Tomorrow, we will be picking colonies and performing PCR on them to determine whether they contain w+zif268.
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We plated the cells, [https://2011.igem.org/Team:Harvard/Protocols#Cultures using this protocol], on spec plates, one of which contained 50 ul of cells and the other 150 ul of cells. The cells have been incubated at 37*, and will be left there overnight. Cells that have taken up the plasmid have spec-resistance and should be able to survive. Tomorrow, we will be picking colonies and performing PCR on them to determine whether they contain w+zif268.
===Team Wolfe===
===Team Wolfe===
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===Team ZF===
===Team ZF===
====PCR to check for omega+zif268====
====PCR to check for omega+zif268====
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All colonies that grew on the plates we left overnight should have spec-resistance, but they may not have the omega and zif268 unit that we inserted [[#Isothermal Assembly|yesterday]] with isothermal assembly. We performed a PCR on 13 colonies picked from the 50 ul plate to find a colony that contains the w+zif268 insertion. We used the same recipe at our PCR reaction [[#PCR of omega+zif268 with overhangs|on Friday]]. If the insert is present, then we should see a band at 709 bp. We also included 2 positive controls: our isothermal assembly product and our selection strain from which the w+zif268 was originally copied.  
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All colonies that grew on the plates we left overnight should have spec-resistance, but they may not have the omega and zif268 unit that we inserted yesterday with isothermal assembly. We performed a PCR on 13 colonies picked from the 50 ul plate to find a colony that contains the w+zif268 insertion. We used the same recipe at our PCR reaction [[#PCR of omega+zif268 with overhangs on Friday. If the insert is present, then we should see a band at 709 bp. We also included 2 positive controls: our isothermal assembly product and our selection strain from which the w+zif268 was originally copied.  
We ran an agarose gel to check the bands:
We ran an agarose gel to check the bands:
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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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==July 11th==
==July 11th==
===Team ZF===
===Team ZF===
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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