Minnesota/9 July 2008

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|1. '''Single Digests''': Performed today, refer to table below.
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|1. '''Single Digests''': Performed today. Incubated digested samples for 2hrs @ 37C. Refer to table below.
 +
 
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{|border="1" align="left"
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|-
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!| Gene !!10x Buffer !!BSA Protein !!H20 !!DNA  !!RE
 +
|-
 +
|Promoter ||5.0uL ||0.5uL ||40.4uL ||2.1uL ||2.0uL, SPE
 +
|-
 +
|LAMBDAcI  ||5.0uL ||0.5uL ||29.5uL ||15.0uL ||1.0uL, XBA 1
 +
|-
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|LAMBDAcI  ||5.0uL ||0.5uL ||29.5uL ||15.0uL ||1.0uL, SPE
 +
|-
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|Terminator ||5.0uL ||0.5uL ||38.0uL ||4.5uL ||2.0uL, XBA 1
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|-
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|}
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|-
 +
|NOTE: RE = Restriction Enzyme
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 +
|-
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|2. '''Ligation''': Performed today. Heat inactivated enzyme @ 65C for 15 min to kill enzymes.Ligate samples. Incubate ligated samples @ 16C for 1hour. Refer to table  
 +
below.
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{|border="1" align="left"
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|-
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!| Genes !!10x Buffer !!H20 !!Base Vector !!Insert  !!DNA Ligase
 +
|-
 +
|LAMBDAcI/Terminator || 2.0uL ||10.7uL ||2.0uL (Term.) ||4.3uL (LAMBDAcI) ||1.0uL
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|-
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|Promoter/LAMBDAcI ||2.0uL ||10.7uL ||2.0uL (Pro.) ||4.3uL (LAMBDAcI) ||1.0uL
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|-
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|}
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|-
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|3. '''Double Digests''': Performed today. After digestion, incubate samples for 2hrs @37C. Heat inactivate restriction site enzymes EcorI and PstI in a water bath @ 65C for 15 minutes to denature the enzymes, thus stopping/controlling digestion. Place digested samples in -20C freezer overnight.
 +
 
 +
{|border="1" align="left"
 +
|-
 +
!| Gene !!10x Buffer !!BSA Protein !!H20 !!DNA  !!RE 1 !!RE 2
 +
|-
 +
|Promoter/LAMBDAcI ||5.0uL ||0.5uL ||22.5uL ||20.0uL ||1.0uL, EcorI ||1.0uL, PstI
 +
|-
 +
|LAMBDAcI/Terminator  ||5.0uL ||0.5uL ||20.0uL ||20.0uL ||1.0uL, EcorI ||1.0uL, PstI
 +
|-
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|Base Vector ||5.0uL ||0.5uL ||15.0uL ||15.0uL ||1.0uL, EcorI ||1.0uL, PstI
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|-
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|}
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 +
|-
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|NOTE: RE = Restriction Enzyme
 +
 
 +
 
 +
|-
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|4. '''Make/Pour Ampicillin Plates'''
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|-
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|5. '''Order Primers for Part Sequencing'''
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|-
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|6. '''Purified Plasmid Prep''': Purified MCherry, Promoter, Terminator and Base Vector because sequencing results were poor.
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|}

Latest revision as of 19:21, 10 July 2008

Back to Notebook Home
Go to Previous Day (July 8)Go to Next Day (July 10)


1. Single Digests: Performed today. Incubated digested samples for 2hrs @ 37C. Refer to table below.
Gene 10x Buffer BSA Protein H20 DNA RE
Promoter 5.0uL 0.5uL 40.4uL 2.1uL 2.0uL, SPE
LAMBDAcI 5.0uL 0.5uL 29.5uL 15.0uL 1.0uL, XBA 1
LAMBDAcI 5.0uL 0.5uL 29.5uL 15.0uL 1.0uL, SPE
Terminator 5.0uL 0.5uL 38.0uL 4.5uL 2.0uL, XBA 1
NOTE: RE = Restriction Enzyme
2. Ligation: Performed today. Heat inactivated enzyme @ 65C for 15 min to kill enzymes.Ligate samples. Incubate ligated samples @ 16C for 1hour. Refer to table

below.

Genes 10x Buffer H20 Base Vector Insert DNA Ligase
LAMBDAcI/Terminator 2.0uL 10.7uL 2.0uL (Term.) 4.3uL (LAMBDAcI) 1.0uL
Promoter/LAMBDAcI 2.0uL 10.7uL 2.0uL (Pro.) 4.3uL (LAMBDAcI) 1.0uL


3. Double Digests: Performed today. After digestion, incubate samples for 2hrs @37C. Heat inactivate restriction site enzymes EcorI and PstI in a water bath @ 65C for 15 minutes to denature the enzymes, thus stopping/controlling digestion. Place digested samples in -20C freezer overnight.
Gene 10x Buffer BSA Protein H20 DNA RE 1 RE 2
Promoter/LAMBDAcI 5.0uL 0.5uL 22.5uL 20.0uL 1.0uL, EcorI 1.0uL, PstI
LAMBDAcI/Terminator 5.0uL 0.5uL 20.0uL 20.0uL 1.0uL, EcorI 1.0uL, PstI
Base Vector 5.0uL 0.5uL 15.0uL 15.0uL 1.0uL, EcorI 1.0uL, PstI
NOTE: RE = Restriction Enzyme


4. Make/Pour Ampicillin Plates
5. Order Primers for Part Sequencing
6. Purified Plasmid Prep: Purified MCherry, Promoter, Terminator and Base Vector because sequencing results were poor.
Retrieved from "http://2008.igem.org/Minnesota/9_July_2008"