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

From 2013.igem.org

(Difference between revisions)
Line 40: Line 40:
<font face="Calibri" size="3">
<font face="Calibri" size="3">
 +
 +
<font size="4"> '''Salt LB recipe''' </font>
 +
 +
<br>
 +
 +
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
 +
 +
<font size="4"> '''Phusion PCR (50ul reaction)''' </font>
<font size="4"> '''Phusion PCR (50ul reaction)''' </font>
Line 53: Line 70:
  0.5 ul Phusion Polymerase
  0.5 ul Phusion Polymerase
 +
Set the PCR machine to run the following cycle
 +
98&deg;C for 02:00
 +
      98&deg;C for 00:30
 +
      65&deg;C for 00:30
 +
      72&deg;C for 04:00
 +
      X35
 +
72&deg;C for 8:00
 +
Hold at 4&deg;C
 +
<br>
<br>
Line 66: Line 92:
  1 ul appropriate diluted template DNA
  1 ul appropriate diluted template DNA
  0.5 ul ''TAQ'' Polymerase
  0.5 ul ''TAQ'' Polymerase
 +
 +
95 for 2
 +
95 for .5
 +
50 for .5
 +
72 for 1
 +
72 for 1
<br>
<br>
 +
 +
Any deviations from these protocols are listed in the notebook when they were used.

Revision as of 21:30, 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
     98°C for 00:30
     65°C for 00:30
     72°C for 04:00
     X35
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.

Retrieved from "http://2013.igem.org/Team:BYU_Provo/Notebook/Cholera_-_Enzymes/Protocols"