Team:Monash Australia/Notebook

From 2010.igem.org

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* Poured 4x SOB Agar plates.
* Poured 4x SOB Agar plates.
-
 
* Poured 3x SOB Agar plates with Streptomycin.
* Poured 3x SOB Agar plates with Streptomycin.
-
 
* Streaked seed stocks of Top 10 cells on SOB Agar and SOB Agar w/ Strep.
* Streaked seed stocks of Top 10 cells on SOB Agar and SOB Agar w/ Strep.
-
 
** Grew at room temp for 48 hours.
** Grew at room temp for 48 hours.
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* Amplified SAM Synthetase from <i>E. coli</i> genomic DNA using designed primers that incorporated biobrick prefix and suffix for RFC10.  
* Amplified SAM Synthetase from <i>E. coli</i> genomic DNA using designed primers that incorporated biobrick prefix and suffix for RFC10.  
-
-> <b>PCR Protocol:</b>
+
<b>PCR Protocol:</b>
-
<table border=0>
+
<table border=1>
<tr>
<tr>
-
  <td>10x Buffer for KOD DNA Pol</td>
+
  <td>10x Buffer for KOD DNA Polymerase</td>
  <td>5 μL</td>
  <td>5 μL</td>
</tr>
</tr>
-
</table>
+
<tr>
 +
<td>25mM MgCl2</td>
 +
<td>3 μL</td>
 +
</tr>
-
** 25mM MgCl2 -
+
<tr>
 +
<td>dNTP's (2 nM each)</td>
 +
<td>5 μL</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>PCR grade water</td>
 +
<td>28.1 μL</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Forward primer (5 pmol/μL)</td>
 +
<td>4 μL</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Reverse primer (5 pmol/μL)</td>
 +
<td>4 μL</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Purified <i>E. coli</i> genomic DNA</td>
 +
<td>0.5 μL</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>KOD DNA Polymerase</td>
 +
<td>0.4 μL</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td><centre>Total</center></td>
 +
<td>50 μL</td>
 +
</tr>
 +
 
 +
</table>
===Date===
===Date===
===Date===
===Date===
===Date===
===Date===

Revision as of 07:18, 22 August 2010

 


Contents

Lab Notebook

09/08/10

Created 1L SOB media using:

  1. 5g yeast extract
  2. 0.5g NaCl
  3. 0.19g KCl
  4. 20g Tryptone

of the 1L of SOB media, 2x 250ml was autoclaved; one was used to make agar medium the other was used for medium. The other 500ml was left for storage, however was subsequently destroyed due to contamination.

3.75g of agar was added to 250ml of SOB media to produce agar media

Created a batch CCMB80 Buffer as followed:

  1. 2.5ml KoAc
  2. 2.95g CaCl2.2H2O
  3. 1g MnCl2.4H2O
  4. 0.5g MgCl2.6H2O
  5. 25ml Glycerol
  6. HCl to bring pH ~ 6.4
  • Poured 4x SOB Agar plates.
  • Poured 3x SOB Agar plates with Streptomycin.
  • Streaked seed stocks of Top 10 cells on SOB Agar and SOB Agar w/ Strep.
    • Grew at room temp for 48 hours.

10/8/10

  • Amplified SAM Synthetase from E. coli genomic DNA using designed primers that incorporated biobrick prefix and suffix for RFC10.

PCR Protocol: 

<td>50 μL</td>
</table>

Date

Date

Date

Retrieved from "http://2010.igem.org/Team:Monash_Australia/Notebook"
10x Buffer for KOD DNA Polymerase 5 μL
25mM MgCl2 3 μL
dNTP's (2 nM each) 5 μL
PCR grade water 28.1 μL
Forward primer (5 pmol/μL) 4 μL
Reverse primer (5 pmol/μL) 4 μL
Purified E. coli genomic DNA 0.5 μL
KOD DNA Polymerase 0.4 μL
<centre>Total</center>