Team:Warsaw/Calendar-Main/22 April 2009

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<ul><li><p>PCR mixture's composition:</p> 2,5&mu;l pfu buffer (Fermentas), 2,5&mu;l MgSO4 (Fermentas), 1,5&mu;l primers, 1,5&mu;l dNTPs (10 mM), 0,5&mu;l pfu turbo polymerase <font color="green">(od Antka, ale KNGiE też powinna działać)</font>, 1&mu;l template DNA from Listeria, solution was topped up with H2O to 25&mu;l.  
<ul><li><p>PCR mixture's composition:</p> 2,5&mu;l pfu buffer (Fermentas), 2,5&mu;l MgSO4 (Fermentas), 1,5&mu;l primers, 1,5&mu;l dNTPs (10 mM), 0,5&mu;l pfu turbo polymerase <font color="green">(od Antka, ale KNGiE też powinna działać)</font>, 1&mu;l template DNA from Listeria, solution was topped up with H2O to 25&mu;l.  
<p>1 repeat of every sample was made (2 different programs).</p>
<p>1 repeat of every sample was made (2 different programs).</p>
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<p>Additionally for each sample was made version with 10X dilution of template.</p>
+
<p>Additionally for each sample was made version with 10&lowast; dilution of template.</p>
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Revision as of 11:16, 12 July 2009


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PCR hly

Kama


Tasks:

  • Amplification of hly

Methods:

  • PCR mixture's composition:

    2,5μl pfu buffer (Fermentas), 2,5μl MgSO4 (Fermentas), 1,5μl primers, 1,5μl dNTPs (10 mM), 0,5μl pfu turbo polymerase (od Antka, ale KNGiE też powinna działać), 1μl template DNA from Listeria, solution was topped up with H2O to 25μl.

    1 repeat of every sample was made (2 different programs).

    Additionally for each sample was made version with 10∗ dilution of template.

  • PCR programs:
  • hly

    4min 95°C 
    (30s 95°C, 35s 42°C, 1min20s 72°C)x3
    (30s 95°C, 35s 47°C, 1min20s 72°C)x28
    10min 72°C
    ~ 7°C

    random program

  • Electrophoretic separation on 1% agarose gel

Results:

  • Gel (from left)
  1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
  2. hly control -
  3. hly
  4. hly (10x diluted template)
  5. hly control -*
  6. hly*
  7. hly (10x diluted template)*

* random program


hly was succesfully amplified (termocykler z gradientem w pokoju 145)