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| [[Melbourne|<Back to team home page>]] | | [[Melbourne|<Back to team home page>]] |
| + | *Gas Vesicle lab notebook: |
| + | ** [[Melbourne/Lab GV Notebook|Gas Vesicles Notebook]] |
| | | |
- | ==Week 1==
| + | *Blue photoreceptor notebook: |
- | *25 June 2007: Prepared LB agar plates Amp & Kana. | + | ** [[Melbourne/Lab BL Notebook|Blue Photoreceptor Notebook]] |
- | *25 June 2007: [[Melbourne/IGEM 2007 kit|Resuspended the following from Registry plates]] & [[Melbourne/Transformation Protocol shorter|Transformed (shorter protocol)]] into competent DH5alpha cells. | + | |
- | *#[[Melbourne/BBa_I15008|'''P2 21A''']] - (BBa_I15008, ho1, Kan) -> No colonies on plates
| + | |
- | *#[[Melbourne/BBa_I15009|'''P2 21C''']] - (BBa_I15009, PcyA, Kan) ->No colonies on plates
| + | |
- | *#[[Melbourne/BBa_R0084|'''P1 11H''']] - (BBa_R0084, OmpR positive promoter, Amp)->Small number of colonies. | + | |
| | | |
- | | + | *Photoreceptor & control system lab notebooks: |
- | | + | ** [[Melbourne/Lab Notebook Weeks 1-4|Weeks 1-4]] |
- | *26 June 2007: [[Melbourne/IGEM 2007 kit|Resuspended the following from Registry plates]] & [[Melb:Transformation Protocol|Transformed]] into Joe's competent DH5alpha cells. | + | ** [[Melbourne/Lab Notebook Weeks 5-8|Weeks 5-8]] |
- | *#[[Melbourne/BBa_B0034|'''P1 3O''']] (BBa_B0034, RBS, Amp) | + | ** [[Melbourne/Lab Notebook Weeks 9-12|Weeks 9-12]] |
- | *#[[Melbourne/BBa_C0051|'''P1 5G''']] (BBa_C0051, c1 protein, Amp) | + | ** [[Melbourne/Lab Notebook Weeks 13-16|Weeks 13-16]] |
- | *#[[Melbourne/BBa_B0010|'''P2 3P''']] (BBa_B0010, Terminator, Amp)
| + | ** [[Melbourne/Lab Notebook Weeks 17-18|Weeks 17-18]] |
- | | + | |
- | *26 June 2007: Streaked the following cells:
| + | |
- | *#[[Melbourne/pJS010|'''pJS010''']] (from solid agar, Amp)
| + | |
- | *#[[Melbourne/Fusion|'''Fusion protein''']] (from glycerol stock, Amp?)
| + | |
- | *#[[Melbourne/BBa_I15010|'''BBa_I15010''']] (from solid agar, Kan)
| + | |
- | | + | |
- | *27 June 2007: [[Melb:Transformation Protocol|Transformed]] into Joe's competent DH5alpha cells.
| + | |
- | *#[[Melbourne/BBa_I15008|'''P2 21A''']] (Kan)
| + | |
- | *#[[Melbourne/BBa_I15009|'''P2 21C''']] (Kan)
| + | |
- | | + | |
- | | + | |
- | ====Liquid culture====
| + | |
- | #[[Melb:Growing up cells|Prepared]] 30mL of Amp LB (100ug/mL). Prepared 5mL Kan LB (50mg/mL).
| + | |
- | #Aliquoted 5mL Amp LB into 6 50mL falcon tubes
| + | |
- | #To the Amp LB aliquots single transformed colonies from plates of the following were introduced:
| + | |
- | #*[[Melbourne/pJS010|'''pJS010''']]
| + | |
- | #*[[Melbourne/Fusion|'''Fusion''']]
| + | |
- | #*[[Melbourne/BBa_B0034|'''P1 3O''']]
| + | |
- | #*[[Melbourne/BBa_C0051|'''P1 5G''']]
| + | |
- | #*[[Melbourne/BBa_B0010|'''P2 3P''']]
| + | |
- | #*[[Melbourne/BBa_R0084|'''P1 11H''']]
| + | |
- | #To the Kan LB a single colony from the transformation plate of [[Melbourne/BBa_I15010|'''BBa_I15010''']] was introduced.
| + | |
- | #Cells incubated at 37degrees with shaking overnight.
| + | |
- | | + | |
- | ===28 June 2007===
| + | |
- | | + | |
- | ====Miniprep====
| + | |
- | #[[Melb:Miniprep protocol|Minipreped]] the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
| + | |
- | #*[[Melbourne/pJS010|'''pJS010''']]
| + | |
- | #*[[Melbourne/Fusion|'''Fusion''']]
| + | |
- | #*[[Melbourne/BBa_B0034|'''P1 3O''']]
| + | |
- | #*[[Melbourne/BBa_C0051|'''P1 5G''']]
| + | |
- | #*[[Melbourne/BBa_B0010|'''P2 3P''']]
| + | |
- | #*[[Melbourne/BBa_R0084|'''P1 11H''']]
| + | |
- | #*[[Melbourne/BBa_I15010|'''I15010''']]
| + | |
- | #Stored in -20 freezer
| + | |
- | | + | |
- | ====Digest====
| + | |
- | | + | |
- | | + | |
- | ====Liquid culture====
| + | |
- | #[[Melb:Growing up cells|Cultured]] the following cells from transformed plates:
| + | |
- | #*[[Melbourne/BBa_R0084|'''P1 11H''']] (BBa_R0084)
| + | |
- | #*[[Melbourne/BBa_I15010|'''BBa_I15010''']]
| + | |
- | #*[[Melbourne/BBa_B0034|'''P1 3O''']] (BBa_B0034)
| + | |
- | #*[[Melbourne/BBa_C0051|'''P1 5G''']] (BBa_C0051)
| + | |
- | #*[[Melbourne/BBa_I15008|'''P1 21A''']] (BBa_I15008)
| + | |
- | #*[[Melbourne/BBa_I15009|'''P1 21C''']] (BBa_I15009)
| + | |
- | | + | |
- | ===29 June 2007===
| + | |
- | ====Miniprep====
| + | |
- | #[[Melb:Miniprep protocol|Minipreped]] the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
| + | |
- | #*[[Melbourne/BBa_R0084|'''P1 11H''']] (BBa_R0084)
| + | |
- | #*[[Melbourne/BBa_I15010|'''BBa_I15010''']]
| + | |
- | #*[[Melbourne/BBa_B0034|'''P1 3O''']] (BBa_B0034)
| + | |
- | #*[[Melbourne/BBa_C0051|'''P1 5G''']] (BBa_C0051)
| + | |
- | #*[[Melbourne/BBa_I15008|'''P1 21A''']] (BBa_I15008)
| + | |
- | #*[[Melbourne/BBa_I15009|'''P1 21C''']] (BBa_I15009)
| + | |
- | | + | |
- | #Stored in -20 freezer
| + | |
- | | + | |
- | ===30 June 2007===
| + | |
- | ==Week 2==
| + | |
- | *2 July 2007: [[Melbourne/IGEM 2007 kit|Resuspended the following from Registry plates]] & [[Melb:Transformation Protocol|Transformed]] into Joe's competent DH5alpha cells.
| + | |
- | *#Q04510
| + | |
- | *#E0241
| + | |
- | *#E0040
| + | |
- | *#B0014
| + | |
- | *#J61035
| + | |
- | | + | |
- | *3 July 2007: [[Melb:Transformation Protocol|Re Transformed]] into Joe's competent DH5alpha cells. Each Plate had additional 40uL of 50mg/ml Ampicillan spread on surface.
| + | |
- | *#[[Melbourne/BBa_J61035|'''P4 8J''']] -> Three colonies -> grew in liquid culture 4 july
| + | |
- | *#[[Melbourne/BBa_E0040|'''P1 5H''']] -> Multiple colonies at edge -> did not grow in overnight liquid culture 4 july 'poor amp spreading'
| + | |
- | *#[[Melbourne/BBa_E0241|'''P2 15L''']] -> Three colonies -> grew in liquid culture 4 july
| + | |
- | | + | |
- | *4 July 2007: | + | |
- | | + | |
- | ====Ampicillin Plates====
| + | |
- | *Made up 25 Ampicillin plates using 400mL LB agar prepared on the 25th of June. | + | |
- | **Used 100mg/ml Ampicillin stock from Gooley Lab - In bag in left freezer near our lab bench.
| + | |
- | | + | |
- | ====Tranformation====
| + | |
- | [[Melbourne/Transformation Protocol|Transformed]] the following and grew on new ampicillin plates | + | |
- | *[[Melbourne/BBa_E0040|'''P1 5H''']]
| + | |
- | *[[Melbourne/BBa_J61035|'''P4 8J''']]
| + | |
- | *[[Melbourne/BBa_E0241|'''P2 15L''']]
| + | |
- | **Also Plated an untransformed control (subjected to transformation protocol in absense of DNA) and a blank plate control (no cells)
| + | |
- | | + | |
- | ====Liquid Culture====
| + | |
- | *[[Melbourne/Growing up cells|Cultured]] 2 colonies from each of the following transformed plates and labelled as follows
| + | |
- | **[[Melbourne/BBa_B0010|'''P2 3P 1''']]
| + | |
- | **[[Melbourne/BBa_B0010|'''P2 3P 2''']]
| + | |
- | **[[Melbourne/BBa_I15010|'''I15010 1''']]
| + | |
- | **[[Melbourne/BBa_I15010|'''I15010 2''']]
| + | |
- | **[[Melbourne/pJS010|'''pJS010 1''']]
| + | |
- | **[[Melbourne/pJS010|'''pJS010 2''']]
| + | |
- | **[[Melbourne/Fusion|'''Fusion 1''']]
| + | |
- | **[[Melbourne/Fusion|'''Fusion 2''']]
| + | |
- | **[[Melbourne/BBa_J61035|'''P4 8J 1''']]
| + | |
- | **[[Melbourne/BBa_J61035|'''P4 8J 2''']]
| + | |
- | **[[Melbourne/BBa_E0241|'''P2 15L 1''']]
| + | |
- | **[[Melbourne/BBa_E0241|'''P2 15L 2''']]
| + | |
- | **[[Melbourne/BBa_Q04510|'''P2 13K 1''']]
| + | |
- | **[[Melbourne/BBa_Q04510|'''P2 13K 2''']]
| + | |
- | **[[Melbourne/BBa_B0014|'''P1 1G 1''']]
| + | |
- | **[[Melbourne/BBa_B0014|'''P1 1G 2''']]
| + | |
- | **[[Melbourne/BBa_R0084|'''P1 11H 1''']]
| + | |
- | **[[Melbourne/BBa_R0084|'''P1 11H 2''']]
| + | |
- | **[[Melbourne/BBa_E0040|'''P1 5H 1''']]
| + | |
- | **[[Melbourne/BBa_E0040|'''P1 5H 2''']]
| + | |
- | **suspected contaminating colony from [[Melbourne/BBa_J61035|'''P4 8J''']]
| + | |
- | | + | |
- | ===5 July 2007===
| + | |
- | | + | |
- | ====Miniprep====
| + | |
- | *1mL culture from the 4th of Julyput aside for glycerol stocks under sterile conditions. Labelled with todays date 5/7
| + | |
- | *miniprepped the following overnight cultures set up on the 4th of July. Final elution was performed with TE buffer prepared on 25/06/07 rather than nuclease free water. Labelled with todays date 5/7.
| + | |
- | **[[Melbourne/BBa_B0010|'''P2 3P 1''']]
| + | |
- | **[[Melbourne/BBa_B0010|'''P2 3P 2''']]
| + | |
- | **[[Melbourne/BBa_I15010|'''I15010 1''']]
| + | |
- | **[[Melbourne/BBa_I15010|'''I15010 2''']]
| + | |
- | **[[Melbourne/pJS010|'''pJS010 1''']]
| + | |
- | **[[Melbourne/pJS010|'''pJS010 2''']]
| + | |
- | **[[Melbourne/Fusion|'''Fusion 1''']]
| + | |
- | **[[Melbourne/Fusion|'''Fusion 2''']]
| + | |
- | **[[Melbourne/BBa_J61035|'''P4 8J 1''']]
| + | |
- | **[[Melbourne/BBa_J61035|'''P4 8J 2''']]
| + | |
- | **[[Melbourne/BBa_E0241|'''P2 15L 1''']]
| + | |
- | **[[Melbourne/BBa_E0241|'''P2 15L 2''']]
| + | |
- | **[[Melbourne/BBa_Q04510|'''P2 13K 1''']](eluted in 130uL due to accidental double application of 50uL elution)
| + | |
- | **[[Melbourne/BBa_Q04510|'''P2 13K 2''']]
| + | |
- | **[[Melbourne/BBa_B0014|'''P1 1G 1''']]
| + | |
- | **[[Melbourne/BBa_B0014|'''P1 1G 2''']]
| + | |
- | *the following liquid cultures were not miniprepped due to failure (no growth)
| + | |
- | **[[Melbourne/BBa_R0084|'''P1 11H 1''']]
| + | |
- | **[[Melbourne/BBa_R0084|'''P1 11H 2''']]
| + | |
- | **[[Melbourne/BBa_E0040|'''P1 5H 1''']]
| + | |
- | **[[Melbourne/BBa_E0040|'''P1 5H 2''']]
| + | |
- | **suspected contaminating colony from [[Melbourne/BBa_J61035|'''P4 8J''']]
| + | |
- | | + | |
- | ====Digest====
| + | |
- | Performed the following [[Melbourne/Diagnostic Digest|digests]] on DNA from the above miniprep
| + | |
- | =====EcoRI/PstI with buffer 3=====
| + | |
- | *[[Melbourne/BBa_I15010|'''I15010 1''']]
| + | |
- | *[[Melbourne/BBa_I15010|'''I15010 2''']]
| + | |
- | *[[Melbourne/BBa_J61035|'''P4 8J 1''']]
| + | |
- | *[[Melbourne/BBa_J61035|'''P4 8J 2''']]
| + | |
- | *[[Melbourne/BBa_E0241|'''P2 15L 1''']]
| + | |
- | *[[Melbourne/BBa_E0241|'''P2 15L 2''']]
| + | |
- | *[[Melbourne/BBa_Q04510|'''P2 13K 1''']]
| + | |
- | *[[Melbourne/BBa_Q04510|'''P2 13K 2''']]
| + | |
- | =====EcoRI/HaeII in buffer 2=====
| + | |
- | *[[Melbourne/BBa_B0010|'''P2 3P 1''']]
| + | |
- | *[[Melbourne/BBa_B0010|'''P2 3P 2''']]
| + | |
- | *[[Melbourne/BBa_B0014|'''P1 1G 1''']]
| + | |
- | *[[Melbourne/BBa_B0014|'''P1 1G 2''']]
| + | |
- | =====XbaI/SpeI in buffer 2=====
| + | |
- | *[[Melbourne/pJS010|'''pJS010 1''']]
| + | |
- | *[[Melbourne/pJS010|'''pJS010 2''']]
| + | |
- | | + | |
- | Incubated for 2hours at 37 degrees, before addition of loading dye and storage at -20
| + | |
- | | + | |
- | ====Transformation====
| + | |
- | *[[Melbourne/Transformation Protocol|Transformed]] P1 11H from resuspended DNA.
| + | |
- | | + | |
- | ====Liquid Culture====
| + | |
- | *[[Melbourne/BBa_E0040|'''P1 5H 1''']]
| + | |
- | *[[Melbourne/BBa_E0040|'''P1 5H 2''']]
| + | |
- | *[[Melbourne/BBa_J61035|'''P4 8J 1''']]
| + | |
- | *[[Melbourne/BBa_J61035|'''P4 8J 2''']]
| + | |
- | *[[Melbourne/BBa_E0241|'''P2 15L 1''']]
| + | |
- | *[[Melbourne/BBa_E0241|'''P2 15L 2''']]
| + | |
- | | + | |
- | ===6 July 2007===
| + | |
- | | + | |
- | ====Digest Gel====
| + | |
- | * Prepared 20 lane 100mL [[Melbourne/Preparing an agarose gel|agarose gel]] with 0.5xTBE buffer.
| + | |
- | *[[Melbourne/Loading a DNA gel|Loaded]] 20uL of digest samples in the following lane order
| + | |
- | *#1kb+ ladder
| + | |
- | *#[[Melbourne/BBa_I15010|'''I15010 1''']]
| + | |
- | *#[[Melbourne/BBa_I15010|'''I15010 2''']]
| + | |
- | *#[[Melbourne/BBa_Q04510|'''P2 13K 1''']]
| + | |
- | *#[[Melbourne/BBa_Q04510|'''P2 13K 2''']]
| + | |
- | *#[[Melbourne/BBa_E0241|'''P2 15L 1''']]
| + | |
- | *#[[Melbourne/BBa_E0241|'''P2 15L 2''']]
| + | |
- | *#[[Melbourne/BBa_J61035|'''P4 8J 1''']]
| + | |
- | *#[[Melbourne/BBa_J61035|'''P4 8J 2''']]
| + | |
- | *#[[Melbourne/BBa_B0014|'''P1 1G 1''']]
| + | |
- | *#[[Melbourne/BBa_B0014|'''P1 1G 2''']]
| + | |
- | *#[[Melbourne/BBa_B0010|'''P2 3P 1''']]
| + | |
- | *#[[Melbourne/BBa_B0010|'''P2 3P 2''']]
| + | |
- | *#[[Melbourne/pJS010|'''pJS010 1''']]
| + | |
- | *#[[Melbourne/pJS010|'''pJS010 2''']]
| + | |
- | *Ran for 1.5hours at 95V
| + | |
- | | + | |
- | ====Miniprep====
| + | |
- | *Put aside 1mL of liquid cultures set up on the 5th for glycerol stocks and labelled with todays date 6/7
| + | |
- | *Miniprepped the remains of the cultures and labelled with todays date 6/7. Samples were eluted with TE buffer.
| + | |
- | *[[Melbourne/BBa_E0040|'''P1 5H 1''']]
| + | |
- | *[[Melbourne/BBa_E0040|'''P1 5H 2''']]
| + | |
- | *[[Melbourne/BBa_J61035|'''P4 8J 1''']]
| + | |
- | *[[Melbourne/BBa_J61035|'''P4 8J 2''']]
| + | |
- | *[[Melbourne/BBa_E0241|'''P2 15L 1''']]
| + | |
- | *[[Melbourne/BBa_E0241|'''P2 15L 2''']]
| + | |
- | | + | |
- | | + | |
- | ====Digest====
| + | |
- | *[[Melbourne/Diagnostic Digest|Digested]] 5uL of each of the above miniprep DNA with EcoRI and PstI in buffer 3.
| + | |
- | *Incubated for 2hours 25min at 37degrees
| + | |
- | *Added 5uL 6x loading dye and stored at -20
| + | |
- | | + | |
- | ====Glycerol Stocks====
| + | |
- | The following [[Melbourne/Glycerol Stocks|glycerol stocks]] were made:
| + | |
- | *Put aside from cultures 5/7 (labelled with this date) | + | |
- | *#[[Melbourne/BBa_E0241|'''P2 15L 1''']] | + | |
- | *#[[Melbourne/BBa_E0241|'''P2 15L 2''']]
| + | |
- | *#[[Melbourne/BBa_J61035|'''P4 8J 1''']]
| + | |
- | *#[[Melbourne/BBa_J61035|'''P4 8J 2''']]
| + | |
- | *#[[Melbourne/BBa_B0014|'''P1 1G 1''']]
| + | |
- | *#[[Melbourne/BBa_B0014|'''P1 1G 2''']]
| + | |
- | *Put aside from cultures 6/7 (labelled with this date)
| + | |
- | *#[[Melbourne/BBa_E0241|'''P2 15L 1''']]
| + | |
- | *#[[Melbourne/BBa_E0241|'''P2 15L 2''']]
| + | |
- | *#[[Melbourne/BBa_J61035|'''P4 8J 1''']]
| + | |
- | *#[[Melbourne/BBa_J61035|'''P4 8J 2''']]
| + | |
- | *#[[Melbourne/BBa_E0040|'''P1 5H 1''']]
| + | |
- | *#[[Melbourne/BBa_E0040|'''P1 5H 2''']]
| + | |
- | Stored at -80
| + | |
- | | + | |
- | ====Liquid Culture====
| + | |
- | [[Melbourne/Growing up cells|Cultured]] the following in 5mL LB
| + | |
- | *[[Melbourne/BBa_I15010|'''I15010 1''']] (Kan)
| + | |
- | *[[Melbourne/BBa_I15010|'''I15010 2''']]
| + | |
- | *[[Melbourne/BBa_R0084|'''P1 11H 1''']] (Amp)
| + | |
- | *[[Melbourne/BBa_R0084|'''P1 11H 2''']]
| + | |
- | | + | |
- | Also placed Kan plate in the incubator to test antibiotic efficiency - no growth on 8/7
| + | |
- | | + | |
- | ===7 July 2007===
| + | |
- | | + | |
- | *Made 10x TAE buffer
| + | |
- | | + | |
- | ====Digest Gel====
| + | |
- | *Prepared 8 lane 60mL [[Melbourne/Preparing an agarose gel|agarose gel]] with 1xTAE buffer.
| + | |
- | *[[Melbourne/Loading a DNA gel|Loaded]] 20uL of digest samples from 6/7 in the following lane order. | + | |
- | *#[[Melbourne/BBa_J61035|'''P4 8J 1''']] | + | |
- | *#[[Melbourne/BBa_J61035|'''P4 8J 2''']]
| + | |
- | *#[[Melbourne/BBa_E0040|'''P1 5H 1''']]
| + | |
- | *#[[Melbourne/BBa_E0040|'''P1 5H 2''']]
| + | |
- | *#[[Melbourne/BBa_E0241|'''P2 15L 1''']]
| + | |
- | *#[[Melbourne/BBa_E0241|'''P2 15L 2''']]
| + | |
- | | + | |
- | ====Glycerol Stocks====
| + | |
- | The following [[Melbourne/Glycerol Stocks|glycerol stocks]] were made and dated 7/7:
| + | |
- | *[[Melbourne/BBa_I15010|'''I15010 1''']]
| + | |
- | *[[Melbourne/BBa_I15010|'''I15010 2''']]
| + | |
- | *[[Melbourne/BBa_R0084|'''P1 11H 1''']]
| + | |
- | *[[Melbourne/BBa_R0084|'''P1 11H 2''']]
| + | |
- | Stored at -80
| + | |
- | | + | |
- | ====Miniprep====
| + | |
- | *Miniprepped the remains of the cultures and labelled with todays date 7/7. Samples were eluted with TE buffer.
| + | |
- | | + | |
- | ====Digest====
| + | |
- | *[[Melbourne/Diagnostic Digest|Digested]] 5uL of each of the above miniprep DNA
| + | |
- | **EcoRI/PstI in buffer 3
| + | |
- | ***[[Melbourne/BBa_I15010|'''I15010 1''']] (Kan)
| + | |
- | ***[[Melbourne/BBa_I15010|'''I15010 2''']]
| + | |
- | ***[[Melbourne/BBa_R0084|'''P1 11H 1''']] (Amp)
| + | |
- | ***[[Melbourne/BBa_R0084|'''P1 11H 2''']]
| + | |
- | *Incubated for 3hours at 37degrees
| + | |
- | *Added 5uL 6x loading dye and stored at -20
| + | |
- | | + | |
- | ===8 July 2007===
| + | |
- | | + | |
- | ====Transformation====
| + | |
- | Resuspended and [[Melbourne/Transformation Protocol|transformed]] the following
| + | |
- | *[[Melbourne/BBa_R0082|'''P1 15P''']](BBa_R0082, Omp R+, Amp) | + | |
- | *[[Melbourne/BBa_R0083|'''P1 17H''']](BBa_R0083, truncated BBa_R0082 Omp R+, Amp) | + | |
- | *[[Melbourne/BBa_E0430|'''P1 11A''']](BBa_E0430, EYFP(RBS+,LVA-,term) Amp)
| + | |
- | *[[Melbourne/BBa_E0840|'''P1 16E''']](BBa_E0430; RBS,GFP,term; Amp)
| + | |
- | The following was also retransformed due to colonies on previous plate appearing to be contaminants and failure of plasmid isolation from these colonies.
| + | |
- | *[[Melbourne/BBa_Q04510|'''P2 13K''']] (BBa_Q04510, c1 inverter, Kan)
| + | |
- | | + | |
- | ==Week 3==
| + | |
- | ===9 July 2007===
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- | *Digested I15010(E/P),P1-11H(E/H),P2-15L 1.5hrs run
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- | *liquid culture P1-11H,I15010,P1-15P,P1-16E,P1-17H,P2-13K
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- | ===10 July 2007===
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- | *Miniprep
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- | *Digest P1-16E,P1-11A,P2-13K,I15010,P1-15P,P1-11H,P1-17H 1.0hrs run
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- | *liquid culture I15010, P2
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- | ===11 July 2007===
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- | *Digest for ligation P1-15P(1)10/7,P1-11H 10/7,P1-17H(1)10/7,P1-16E(2)10/7,P1-11A(1)10/7
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- | *loaded order X/P P1-11A,X/P P1-16E,S/P P1-15P,S/P P1-11H
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- | ** Spe1(6uL)/Pst1(7.5uL)/AP(1.5uL), Buffer2 9uL, BSA 9uL, milliQ 27uL into 20uL aliquots with 10uL DNA.
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- | ** Xbal1(3uL)/PstI(4uL),Buffer2 6uL,10XBSA 6uL,milliQ 21uL into 20uL aliquots with 10uL DNA
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- | *Excise bands of interest and purify invitrogen
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- | *liquid culture P1-11A,P1-15P 10ml
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- | *Transform P2-21B,P2-23N,P3-20I into DB3.1 heat shock.
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- | *Glycerol stocks P1-11A,P1-16E,P1-11H,P1-15P,P1-17H
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- | ===12 July 2007===
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- | *Ran Gel P1-11A,P1-16E,P1-15P,P1-11H
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- | *miniprepped P1-11A,P1-15P
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- | *Digest
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- | *Ligate control=(2uL ligase buffer,1uL ligase,5uL vector(P1-15P),12uL H20)
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- | *Ligate (2uL ligase buffer,1uL ligase,5uL vector(P1-15P),10uL insert(P1-11A),2uL H20)
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- | *Liquid culture transformants 11/7
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- | ===13 July 2007===
| + | |
- | *miniprep cultures from transformants 11/7
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- | *Digestion P1-15P and P1-11A from 12/7/07 37degC 3hours 15 minutes stopped 5uL of 6X loading dye.
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- | *Excise bands 800bp from P1-11A, 2Kbp from P1-15P and purified.
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- | *Glycerol stocks of P3-20I,P2-21B,P2-23N
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- | *Transform DH5a with ligation product
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- | ===14 July 2007===
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- | *Transform using 10uL of ligation reaction
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- | ==Week 4==
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- | ===16 July 2007===
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- | *[[Melbourne/colony pcr|Colony PCR]]
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- | ===17 July 2007===
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- | ===18 July 2007===
| + | |
- | *Purchased XbaI,EcoRI,PstI
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- | *Miniprepped cultures 1,3,6 from 16/7
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- | *Digestion with E/P
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- | *Ran Gel:std,ctrl(from PCR reaction 1 earlier),Digest1,3,6,(P2-15P)ages ago,(P1-11A)ages ago?
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- | | + | |
- | ===19 July 2007===
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- | ===20 July 2007===
| + | |
- | ==Week 5==
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- | ===23 July 2007===
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- | ===24 July 2007===
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- | ===25 July 2007===
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- | ===26 July 2007===
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- | ===27 July 2007===
| + | |
- | ==Week 6==
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- | ===30 July 2007===
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- | ===31 July 2007===
| + | |
- | ===1 Aug 2007===
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- | ===2 Aug 2007===
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- | ===3 Aug 2007===
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