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

From 2013.igem.org

(Difference between revisions)
Line 73: Line 73:
*98°C for 02:00
*98°C for 02:00
Repeat the next three steps 35X
Repeat the next three steps 35X
-
**98°C for 00:30
+
* 98°C for 00:30
-
**65°C for 00:30
+
* 65°C for 00:30
-
**72°C for 04:00
+
* 72°C for 04:00
*72°C for 8:00
*72°C for 8:00
Hold at 4°C
Hold at 4°C

Revision as of 21:42, 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

  • 98°C for 02:00

Repeat the next three steps 35X

  • 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

95 for 2 95 for .5 50 for .5 72 for 1 72 for 1


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