Team:BYU Provo/Notebook/Cholera - Enzymes/Protocols

From 2013.igem.org

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<font size="4"> '''PCR Product Restriction Digest(50ul reaction)''' </font>
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<font size="4"> '''Restriction Digest (50ul reaction)''' </font>
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*30 ul DNA sample
*30 ul DNA sample
*2 ul each restriction enzyme
*2 ul each restriction enzyme
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Place in 37&deg;C incubator or water bath for 1.5 hours
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<font size="4"> '''Ligation''' </font>
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For each ligation reaction add
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*6.5 ul H2O
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*1.5 ul 10X ligase buffer
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*1 ul T4 DNA ligase
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*3 ul vector
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*3 ul insert
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Incubate the reaction at room temperature for 30 minutes
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<font size="4"> '''Transformations''' </font>
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Set two heat blocks at 42&deg;C and 37&deg;C respectively. Thaw 25 ul of chemically competent cells on ice and add 5 ul of DNA to be cloned in. Place on ice for approximately 5 minutes. Heat shock at 42&deg;C for 1 minute and immediately place back on ice for 2-5 minutes. Add 500 ul LB to the reaction and incubate at 37&deg;C for 30 minutes. Plate 100 ul of cell on appropriate antibiotic plate and incubate at 37&deg;C overnight.
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<font size="4"> '''''V. cholerae'' Biofilm Degradation Assay''' </font>
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For each sample add 1 ml salt LB and 50 ul overnight seed. Add appropriate
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===Any deviations from these protocols are listed in the notebook when they were used===
===Any deviations from these protocols are listed in the notebook when they were used===

Revision as of 22:13, 27 September 2013


Cholera - Enzyme Laboratory Protocols



Cholera Enzyme
March-April
May-June
July-August
September-October
Protocols

Cholera-Enzyme Protocols



Salt LB recipe


To 950 ml distilled H2O add

  • 24.00 g NaCl
  • 11.90 g MgCl2-6H2O
  • 02.00 g CaCl2-2H2O
  • 00.85 g KCl

Adjust pH to 7.8
Add

  • 10.00 g Bacto-tryptone
  • 05.00 g Yeast extract

Fill to 1000 ml and autoclave


Phusion PCR (50ul reaction)


For each sample add:

  • 35 ul ddH2O
  • 10 ul 5X Phusion buffer
  • 1.5 ul 10mM dNTP's
  • 1 ul each Primer (50 uM stock)
  • 1 ul appropriate diluted template DNA
  • 0.5 ul Phusion Polymerase

Set the PCR machine to run the following cycle with the middle three phases being repeated 35 times

  • 98°C for 02:00
  • 98°C for 00:30
  • 65°C for 00:30
  • 72°C for 04:00
  • 72°C for 8:00

Hold at 4°C


Taq polymerase PCR (50ul reaction)


For each sample add:

  • 40 ul ddH2O
  • 5 ul 10X TAQ buffer
  • 1.5 ul 10 mM dNTP's
  • 1 ul each Primer (50 uM stock)
  • 1 ul appropriate diluted template DNA
  • 0.5 ul TAQ Polymerase

Set the PCR machine to run the following cycle with the middle three phases being repeated 35 times

  • 95 for 2:00
  • 95 for 0:30
  • 50 for 0:30
  • 72 for 1:00
  • 72 for 1:00

Hold at 4°C


Restriction Digest (50ul reaction)


For each sample add:

  • 14 ul ddH2O
  • 5 ul 10X NEB buffer
  • 0.5 ul 100X BSA
  • 30 ul DNA sample
  • 2 ul each restriction enzyme

Place in 37°C incubator or water bath for 1.5 hours


Ligation


For each ligation reaction add

  • 6.5 ul H2O
  • 1.5 ul 10X ligase buffer
  • 1 ul T4 DNA ligase
  • 3 ul vector
  • 3 ul insert

Incubate the reaction at room temperature for 30 minutes


Transformations


Set two heat blocks at 42°C and 37°C respectively. Thaw 25 ul of chemically competent cells on ice and add 5 ul of DNA to be cloned in. Place on ice for approximately 5 minutes. Heat shock at 42°C for 1 minute and immediately place back on ice for 2-5 minutes. Add 500 ul LB to the reaction and incubate at 37°C for 30 minutes. Plate 100 ul of cell on appropriate antibiotic plate and incubate at 37°C overnight.


V. cholerae Biofilm Degradation Assay


For each sample add 1 ml salt LB and 50 ul overnight seed. Add appropriate


===Any deviations from these protocols are listed in the notebook when they were used===

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