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

From 2013.igem.org

(Difference between revisions)
Line 70: Line 70:
*0.5 ul Phusion Polymerase
*0.5 ul Phusion Polymerase
-
Set the PCR machine to run the following cycle
+
Set the PCR machine to run the following cycle with the middle three being repeated 35 times
*98°C for 02:00
*98°C for 02:00
-
 
*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
-
X35
 
*72°C for 8:00
*72°C for 8:00
Hold at 4°C
Hold at 4°C
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*0.5 ul ''TAQ'' Polymerase
*0.5 ul ''TAQ'' Polymerase
-
95 for 2
+
*95 for 2:00
-
95 for .5
+
*95 for 0:30
-
50 for .5
+
*50 for 0:30
-
72 for 1
+
*72 for 1:00
-
72 for 1
+
*72 for 1:00
<br>
<br>
===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 21:47, 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 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
  • 95 for 2:00
  • 95 for 0:30
  • 50 for 0:30
  • 72 for 1:00
  • 72 for 1:00


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