Minnesota/23 June 2008

From 2008.igem.org

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[[Image:6.23gel.jpg|700px||center|thumb|A gel run on 6/23]]
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{|style="align:left" width="965"
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|'''[[Team:Minnesota/NotebookComparator|  Back to Notebook Home]]'''
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|-
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|'''[[Minnesota/20 June 2008|Go to Previous Day (June 20)]]'''|| width=158|'''[[Minnesota/24 June 2008|Go to Next Day (June 24)]]'''
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|}
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{|
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|1. '''Continued plasmid prep work'''
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|2. '''Look up available Biobrick Plasmids'''
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|-
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|3. '''Begin iGEM wiki'''
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|-
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|4.    ''' Transformation Results:'''
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|a. The transformations from 6-19 were successful for all but two of the BioBrick parts. The two unsuccessful BioBricks were those with kanamycin resistance - these were looked into to ensure that they did indeed have kanamycin resistance and the antibiotic was correct.
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|b. The resulting cultures cultures were miniprepped to isolate plasmids.
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|-
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|5. '''Biobrick parts were punched out''' of the notebook provided by the registry. The parts were carved out of the book with a straight edge razor. This blade was cleaned between each BioBrick by dipping in strong bleach solution, sterile H20, and 100% ethanol. Paper punches were placed in sterile 1.5mL eppendorf tubes for transport back from St. Paul. 5uL TE (ph 8.0) was added to each and they were placed in a 50 degree Celsius bath for 20 minutes. Transformations into TOP10 E.Coli cells were carved out with each of these BioBricks by following the iGEM transformation steps. The solutions recovered in 250uL of SOC medium. 100uL of each of these solutions were plated on LB plates containing the appropriate antibiotic after the 2 hour recovery preiod. These plates were allowed to grow overnight in an incubator @ 37C. The following parts were punched out:
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|a. pSB 4A5 - a low-copy base plasmid with ampicillin resistance.
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|b. pSB 4c5 - a low-copy base plasmid with omoramphenicol resistance.
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|c. pSB 3K5 - a medium-copy base plasmid with kanamycin resistance.
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|d. p22 MNT gene - kanamycin resistant...duplicate of #5
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|e. p22 MNT promoter - kanamycin resistant...duplicate of #4.
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|-
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|6. '''Perform double digest.''' Double digest is when the plasmid is cut at two different restriction sites by using restriction enzymes.
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|}
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{|border="1"
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|-
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!| Double Digest ||4x ||10x
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|-
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| |1.0 micrograms DNA ||2 ||1
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|-
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| |(10x) Buffer 2 ||20 ||50
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|-
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| |(100x) BSA  ||2 ||5
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|-
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| |(20 units/uL) XBA1 ||2 ||5
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|-
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| |(10 units/uL) Spe1 ||4 ||10
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|-
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| |H20        ||164 ||420
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|-
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| |Total sln      ||50 uL ||50 uL
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|}

Latest revision as of 20:39, 2 July 2008

Back to Notebook Home
Go to Previous Day (June 20)Go to Next Day (June 24)
1. Continued plasmid prep work
2. Look up available Biobrick Plasmids
3. Begin iGEM wiki
4. Transformation Results:
a. The transformations from 6-19 were successful for all but two of the BioBrick parts. The two unsuccessful BioBricks were those with kanamycin resistance - these were looked into to ensure that they did indeed have kanamycin resistance and the antibiotic was correct.
b. The resulting cultures cultures were miniprepped to isolate plasmids.
5. Biobrick parts were punched out of the notebook provided by the registry. The parts were carved out of the book with a straight edge razor. This blade was cleaned between each BioBrick by dipping in strong bleach solution, sterile H20, and 100% ethanol. Paper punches were placed in sterile 1.5mL eppendorf tubes for transport back from St. Paul. 5uL TE (ph 8.0) was added to each and they were placed in a 50 degree Celsius bath for 20 minutes. Transformations into TOP10 E.Coli cells were carved out with each of these BioBricks by following the iGEM transformation steps. The solutions recovered in 250uL of SOC medium. 100uL of each of these solutions were plated on LB plates containing the appropriate antibiotic after the 2 hour recovery preiod. These plates were allowed to grow overnight in an incubator @ 37C. The following parts were punched out:
a. pSB 4A5 - a low-copy base plasmid with ampicillin resistance.
b. pSB 4c5 - a low-copy base plasmid with omoramphenicol resistance.
c. pSB 3K5 - a medium-copy base plasmid with kanamycin resistance.
d. p22 MNT gene - kanamycin resistant...duplicate of #5
e. p22 MNT promoter - kanamycin resistant...duplicate of #4.
6. Perform double digest. Double digest is when the plasmid is cut at two different restriction sites by using restriction enzymes.
Double Digest 4x 10x
1.0 micrograms DNA 2 1
(10x) Buffer 2 20 50
(100x) BSA 2 5
(20 units/uL) XBA1 2 5
(10 units/uL) Spe1 4 10
H20 164 420
Total sln 50 uL 50 uL
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