Contents
April
April 1, 2011
Confirmation Gel of I1343, B0034, C0040 & B0015
- Each of I1343, B0034, C0040 & B0015 were restricted following the Restriction Protocol 4.
- A gel was made following the 1% Agarose Gel Protocol (100mL).
- The restrictions were loaded onto the gel with a 1kb ladder and 100bp ladder.
- The gel ran at 120V for 60 minutes.
- The gel was then stained in Ethidium Bromide for 10 minutes.
- Results: The parts were confirmed with the correct sizes.
April 2, 2011
Assembly of I1343 & B0034 and C0040 & B0015
- Each of I1343, B0034, C0040 & B0015 were restricted following the Restriction Protocol 1 where all odd parts are upstream and even parts are downstream.
- I1343 & B0034 were ligated together in pSB1A3 following the Ligation Protocol 1.
- C0040 & B0015 were ligated together in pSB1A3 following the Ligation Protocol 1.
- Both ligations were transformed following the Transformation Protocol 1.
April 21, 2011
April 23, 2011
April 27, 2011
April 28, 2011
- Under Construction
May
May 18, 201
Transformation of P0440 in pSB1A2 from 2010 Kit Plate
- 10µL of Milli-Q water and extraction of P0440 in pSB1A2 from the 2010 kit plate (10I) were mixed together.
- The transformation was done following the Transformation Protocol 1.
May 20, 2011
Miniprep of P0440 in pSB1A2
- Miniprep of P0440 in pSB1A2 was made following the Miniprep Protocol (Modified Bench Protocol: QIAprep Spin Miniprep Kit).
Assembly of Multiple Parts
- Each of I13453, P0440, K331033, K331035, K331035, B0015, K249002, B0015, R0010 & B0034 were restricted where all odd parts were upstream and even parts were downstream.
- I1343 & P0330 were ligated together in pSB1A3 following the Ligation Protocol 1.
- K331033 & K331035 were ligated together in pSB1A3 following the Ligation Protocol 1.
- K331035 & B0015 were ligated together in pSB1A3 following the Ligation Protocol 1.
- K249002 & B0015 were ligated together in pSB1A3 following the Ligation Protocol 1.
- R0010 & B0034 were ligated together in pSB1A3 following the Ligation Protocol 1.
- All ligations and a negative control (water) were transformed following the Transformation Protocol 1.
- Gel
May 26, 2011
Transformation of K325909 in pSB1C3 and K325229 in pSB1C3 from 2011 Kit Plate
- 10µL of Milli-Q water and extraction of K325909 in pSB1C3 from the 2011 kit plate (17m) were mixed together.
- 10µL of Milli-Q water and extraction of K325299 in pSB1C3 from the 2011 kit plate (17c) were mixed together.
- The transformation was done following the Transformation Protocol 1.
June
June 8, 2011
Minipreps of Multiple Parts
- Minipreps of K331022, K331008, K331024, xylE, K331024, K331007, K331022, K331008 & K331007 were done following the Miniprep Protocol (Modified Bench Protocol: QIAprep Spin Miniprep Kit Using Microcentrifuge).
June 9, 2011
Assembly of pBAD-rbs & lumazine-dt
- Each of pBAD-rbs & lumazine-dt were restricted following the Restriction Protocol 1
- pBAD-rbs & lumazine-dt were ligated together in pSB1C3 following the Ligation Protocol 1.
- The ligation was transformed following the Transformation Protocol 1.
June 10, 2011
Transformation of pBAD-P0440
- pBAD-P0440 in pSB1K3 was transformed following the Transformation Protocol 1.
Overnight Cultures of pBAD-rbs & lumazine-dt
- Overnight cultures of pBAD-rbs & lumazine-dt grown following the Overnight Cultures Protocol.
June 11, 2011
Assembly of K331033 & K331035 and R0010 & B0034
- Each of K331033, K331035, R0010 & B0034 were restricted following the Restriction Protocol 1 where all odd parts are upstream and even parts are downstream.
- K331033 & K331035 were ligated together in pSB1K3 following the Ligation Protocol 1.
- R0010 & B0034 were ligated together in pSB1K3 following the Ligation Protocol 1.
- Both ligations were transformed following the Transformation Protocol 1.
June 12, 2011
Overnight Cultures of K331033-K331035 & R0010-B0034
- Overnight cultures of K331033-K331035 & R0010-B0034 grown following the Overnight Cultures Protocol.
June 13, 2011
Assembly of Multiple Parts
- Each of K331033, K331035, R0010, B0034, pBAD-rbs & lumazine-dt were restricted following the Restriction Protocol 1 where all odd parts are upstream and even parts are downstream and with the pSB1K3 destination plasmid.
- K331033 & K331035 were ligated together in pSB1K3 following the Ligation Protocol 1.
- R0010 & B0034 were ligated together in pSB1K3 following the Ligation Protocol 1.
- pBAD-rbs & lumazine-dt were ligated together in pSB1K3 following the Ligation Protocol 1.
- All ligations were transformed following the Transformation Protocol 1.
June 14, 2011
Minipreps of R0010-B0034 & pBAD-rbs-lumazine-dt
- Minipreps of R0010-B0034 & pBAD-rbs-lumazine-dt were made following the Miniprep Protocol (Modified Bench Protocol: QIAprep Spin Miniprep Kit).
Overnight Cultures of K331033-K331035 & R0010-B0034
- Overnight cultures of K331033-K331035 & R0010-B0034 grown following the Overnight Cultures Protocol.
June 15-16, 2011
Assembly of K331033 & K331035 and R0010 & B0034
- Each of K331033, K331035, R0010 & B0034 were restricted following the Restriction Protocol 1 where all odd parts are upstream and even parts are downstream.
- K331033 & K331035 were ligated together in pSB1K3 following the Ligation Protocol 1.
- R0010 & B0034 were ligated together in pSB1K3 following the Ligation Protocol 1.
- Both ligations were transformed following the Transformation Protocol 1.
Confirmation Gel of pBAD-rbs-lumazine-dt & R0010-B0034
- Each of pBAD-rbs-lumazine-dt & R0010-B0034 were restricted following the Restriction Protocol 4.
- A gel was made following the 1% Agarose Gel Protocol (100mL), but 1.5% with 1.50g of Agarose and 100mL of 1x TAE.
- The restrictions were loaded onto the gel with a 1kb ladder and 100bp ladder.
- The gel ran at 100V for 60 minutes.
- The gel was then stained in Ethidium Bromide for 10 minutes.
June 22, 2011
PCR of pBAD-P0440 in pSB1K3
- pBAD-P0440 was PCRed following the PCR Protocol.
June 23, 2011
Confirmation Gel of pBAD-P0440 (PCR)
- pBAD-P0440 (PCR) was restricted following the Restriction Protocol 4.
- A gel was made following the 1% Agarose Gel Protocol (100mL).
- The restrictions were loaded onto the gel with a 1kb ladder.
- The gel ran at 100V for 90 minutes.
- The gel was then stained in Ethidium Bromide for 10 minutes.
- TABLE? PICTURE?
Overnight Cultures of pBAD-rbs-lumazine-dt & K331033-K331035
- Overnight cultures of pBAD-rbs-lumazine-dt & K331033-K331035 grown following the Overnight Cultures Protocol.
June 24, 2011
Minipreps of pBAD-rbs-lumazine-dt & K331033-K331035
- 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
PCR of pBAD-P0440 in pSB1K3 and P0440 was done using PCR protocol Assembly of R0010 with B0034 in pSB1C3 Transformation for pBad-RBS and pBad- P0440 into Dh5a E. Coli
June 30th, 2011
Ran 1% Agarose Gel from June 29 PCR of pBAD-p0440 and p0440
July
July 2, 2011
Picked Colonies and grew overnight
- 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
PCR out inserts from July 03, of pBAD-P0440
- 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
Repeated July 05, but only for pBad p0440 (2,4)
July 7th, 2011
Assembled I13453 with B0034 and into pSB1K3 with J04500
- Assembly done using Restriction, ligation, and transformation techniques
July 11th, 2011
Assembly of pLacI-rbs and Lumazine synthase- dt Plasmid transfer of Ag43
- 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
Assembly of K331033 and K331035
- 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
Ligation of K331033 to K3310335 into pSB1K3
- 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
Assembly of pLacI –Rbs in pSB1C3 and Lumazine Dt in pSB1AK3
- Assembly of pLacI –Rbs in pSB1C3 and Lumazine Dt in pSB1AK3 using restriction digest and gel extraction protocols
July 19th,2011
Assembly of K346007 and J04500 into destination plasmid pSB1C3
- 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
Tried to pre-aliquot restriction
- 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
A Colony PCR was run for Parts K346007 and J04500 to confirm assemblies
- This was done following the colony PCR protocol
July 26th,2011
Picking of colonies
- 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
Assembly of following parts:
- 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
Ran a PCR gel for Justin to confirm PCR insert was correct
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