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Team:Lethbridge/Notebook/Lab Work/Group1
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<i>Minipreps of pBAD-rbs-lumazine-dt & K331033-K331035</i> | <i>Minipreps of pBAD-rbs-lumazine-dt & K331033-K331035</i> | ||
* Minipreps of pBAD-rbs-lumazine-dt & K331033-K331035 were made following the Miniprep Protocol (Modified Bench Protocol: QIAprep Spin Miniprep Kit Using Microcentrifuge). | * Minipreps of pBAD-rbs-lumazine-dt & K331033-K331035 were made following the Miniprep Protocol (Modified Bench Protocol: QIAprep Spin Miniprep Kit Using Microcentrifuge). | ||
| + | |||
| + | == June 29th, 2011== | ||
| + | <i>PCR of pBAD-P0440 in pSB1K3 and P0440 was done using PCR protocol </i> | ||
| + | <i>Assembly of R0010 with B0034 in pSB1C3 </i> | ||
| + | <i> Transformation for pBad-RBS and pBad- P0440 into Dh5a E. Coli</i> | ||
| + | == June 30th, 2011== | ||
| + | <i> Ran 1% Agarose Gel from June 29 PCR of pBAD-p0440 and p0440 </i> | ||
| + | |||
| + | |||
| + | =July= | ||
| + | == July 2, 2011== | ||
| + | <i>Picked Colonies and grew overnight </i> | ||
| + | *4 colonies picked from pBad rbs in pSB1T3 | ||
| + | *3 colonies picked from pBad-p0440 | ||
| + | *picked 3 colonies from 2 plates of pBAD-P0440 in pSB1C3 | ||
| + | **1 colony picked from plate A, and 2 colonies from plate B | ||
| + | |||
| + | == July 03,2011== | ||
| + | *None of the 4 colonies of pBAD rbs grew, so they were re-grown in the incubator | ||
| + | *Samples of pBAD-p0440 were mini prepped and stored in team 1 DNA box | ||
| + | *Picked 3 samples of pBad- p0440 in psb1C3 and 3 samples of pBAD p0440 in pSB1K3 | ||
| + | |||
| + | == July 05,2011== | ||
| + | <i> PCR out inserts from July 03, of pBAD-P0440</i> | ||
| + | *PCR out inserts from July 03, of pBAD-P0440 samples using prefix and antisense suffix primers and run on agarose gel at 120V to determine if plasmid contains the correct insert, using po440 as a control. | ||
| + | |||
| + | == July 06,2011== | ||
| + | <i>Repeated July 05, but only for pBad p0440 (2,4)</i> | ||
| + | |||
| + | == July 7th, 2011== | ||
| + | <i>Assembled I13453 with B0034 and into pSB1K3 with J04500</i> | ||
| + | *Assembly done using Restriction, ligation, and transformation techniques | ||
| + | |||
| + | == July 11th, 2011== | ||
| + | <i>Assembly of pLacI-rbs and Lumazine synthase- dt</i> | ||
| + | <i>Plasmid transfer of Ag43</i> | ||
| + | *Assembled pLacI-rbs and Lumazine synthase- dt into destination plasmid pSB1C3, followed restriction digest, ligation, and transformation procedures. | ||
| + | *Plasmid Transfer of Ag43 from pSB1C3 to pSB1K3 using cut sites EcoRI and PstI | ||
| + | *Ran a 1% Agarose Gel using 0.5 TAE buffer solution for parts BBa-J04500 in pSB1AK3, R0040, and Lumazine synthase-Dt to confirm sizes. Used Restriction procedure, cutting with EcoRI | ||
| + | |||
| + | == July 13th, 2011== | ||
| + | <i>Assembly of K331033 and K331035</i> | ||
| + | *Did a restriction and Gel Extraction for K331033 cutting at EcoRI and SpeI, K331035 cutting at PstI and XbaI, and pSB1K3 cutting at EcoRI and PstI | ||
| + | *Ran the gel for 80 minutes at 120Volts | ||
| + | *Used Transformation protocol to Transformed XylE, XylF, XylT, XylK, XylQ, into Dh5a E. Coli cells | ||
| + | |||
| + | == July 14, 2011== | ||
| + | <i>Ligation of K331033 to K3310335 into pSB1K3</i> | ||
| + | *Performed a ligation of K331033 to K3310335 into pSB1K3 and transformation of ligated parts into Dh5a E. Coli. Used Transformation and ligation protocols. Plated culture on Kanmycin plates | ||
| + | |||
| + | ==July 15th, 2011== | ||
| + | <i> Assembly of pLacI –Rbs in pSB1C3 and Lumazine Dt in pSB1AK3</i> | ||
| + | *Assembly of pLacI –Rbs in pSB1C3 and Lumazine Dt in pSB1AK3 using restriction digest and gel extraction protocols | ||
| + | |||
| + | == July 19th,2011== | ||
| + | <i> Assembly of K346007 and J04500 into destination plasmid pSB1C3</i> | ||
| + | *Assembly of K346007 and J04500 into destination plasmid pSB1C3 parts were restricted, ligated and transformed using appropriate protocols. | ||
| + | *Results: all of the plates had significant bacterial growth, will repeat experiment using LB instead of SOC during transformation | ||
| + | |||
| + | == July 20th,2011== | ||
| + | <i>Tried to pre-aliquot restriction</i> | ||
| + | *Created a master-mix from appropriately sized aliquots that can be stored in the -20 freezer. Two mixes were created in the following volumes: | ||
| + | **16micro liters miliQ H2O | ||
| + | **2.5 micro liters Buffer II | ||
| + | **0.5 micro liters BSA 100X | ||
| + | **0.5 micro liters EcoRI | ||
| + | **0.5 micro liters SpeI | ||
| + | **20 micro liters total restriction mixture | ||
| + | |||
| + | == July 25th, 2011== | ||
| + | <i>A Colony PCR was run for Parts K346007 and J04500 to confirm assemblies</i> | ||
| + | * This was done following the colony PCR protocol | ||
| + | |||
| + | == July 26th,2011== | ||
| + | <i>Picking of colonies</i> | ||
| + | *Cells were picked with parts J04500- K346007 in them, for plasmid extraction by miniprep protocol. 23 colonies were picked in total and placed in 5 ml of lb in test tubes, along with 5micro liters of chloramphenicol was added to each test tube. Picked colonies were placed in the shaker overnight at 37 degrees Celsius | ||
| + | |||
| + | == July 27th, 2011== | ||
| + | <i>Assembly of following parts:</i> | ||
| + | **K331033 to K331035 | ||
| + | **J04500 to Lumazine Synthase Dt | ||
| + | **pBad to P0440 | ||
| + | **P0440 to K331033 | ||
| + | **Lumazine synthase dt to pBad | ||
| + | *A restriction was done using the restriction protocol following a Gel extraction protocol. A ligation was performed and contents incubated at room temperature for 30 mins. Controls were run with downstream part being substituted as water. | ||
| + | *Restricted J04500-k346007 with EcoRI, and PstI following restriction protocol, to confirm assembly success, restriction were loaded into a 08% Agarose gel. 23 samples from previous day was loaded into gel. Lanes 16 and 17 showed expected sizes | ||
| + | |||
| + | == July 28th,2011== | ||
| + | <i>Ran a PCR gel for Justin to confirm PCR insert was correct</i> | ||
Revision as of 23:34, 28 September 2011
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