Team:Warsaw/Calendar-Main/1 July 2009

From 2009.igem.org

(Difference between revisions)
Line 15: Line 15:
<p>Methods:</p>
<p>Methods:</p>
<ul><li><p>PCR mixture's composition:</p> 2,5ul pfu buffer (Fermentas), 2,5ul MgSO4 (Fermentas), 1,5ul primers, 1,5ul dNTPs (10 mM), 0,5ul pfu turbo polymerase, 1ul template DNA from Listeria, solution was topped up with H2O to 25ul.  
<ul><li><p>PCR mixture's composition:</p> 2,5ul pfu buffer (Fermentas), 2,5ul MgSO4 (Fermentas), 1,5ul primers, 1,5ul dNTPs (10 mM), 0,5ul pfu turbo polymerase, 1ul template DNA from Listeria, solution was topped up with H2O to 25ul.  
-
<p>One of the samples also contained 1 ul of DMSO</p>
 
</li>
</li>
</ul>
</ul>
<ul>
<ul>
<li>PCR programs:</li>
<li>PCR programs:</li>
-
<p>hly</p><pre align="left">4min 95&deg;C <br/> (30s 95&deg;C, 40s 42-47&deg;C, 1min30s 72&deg;C)x3 <br/> (30s 95&deg;C, 40s 44-54&deg;C, 1min30s 72&deg;C)x28 <br/> 10min 72&deg;C <br/> ~ 7&deg;C</pre>
+
<p>hly</p><pre align="left">4min 95&deg;C <br/> (30s 95&deg;C, 40s 40-48&deg;C, 1min30s 72&deg;C)x3 <br/> (30s 95&deg;C, 40s 44-55&deg;C, 1min30s 72&deg;C)x28 <br/> 10min 72&deg;C <br/> ~ 7&deg;C</pre>
</ul>
</ul>
<ul>
<ul>

Revision as of 14:33, 5 July 2009


Previous day
return to main notebook page
Previous entry
next notebook entry

 



Gradient PCR Llo

Kuba


Tasks:

  • Amplification of llo(hly) for fusion with the secretion signal peptide

Methods:

  • PCR mixture's composition:

    2,5ul pfu buffer (Fermentas), 2,5ul MgSO4 (Fermentas), 1,5ul primers, 1,5ul dNTPs (10 mM), 0,5ul pfu turbo polymerase, 1ul template DNA from Listeria, solution was topped up with H2O to 25ul.
  • PCR programs:
  • hly

    4min 95°C 
    (30s 95°C, 40s 40-48°C, 1min30s 72°C)x3
    (30s 95°C, 40s 44-55°C, 1min30s 72°C)x28
    10min 72°C
    ~ 7°C
  • Electrophoretic separation on 1% agarose gel

Results:

  • Gel (from left)
  1. M - GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
  2. 1-5 - samples (annealing temperature increases to from the left to the right)

  3. many unspecific products were obtained (product of the correct lenght marked with an arrow)


    Quality check (cro PCR product and pKS isolate)



    Kuba



    Cro(wstawki)%2Bpks(test).JPG

    • Gel (from the left)
    1. CRO
    2. pKS isolate
    3. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)




    April
    MTWTFSS
        1 2 3 4 5
    6 7 8 9 10 11 12
    13 14 15 16 17 18 19
    20 21 22 23 24 25 26
    27 28 29 30
    May
    MTWTFSS
            1 2 3
    4 5 6 7 8 9 10
    11 12 13 14 15 16 17
    18 19 20 21 22 23 24
    25 26 27 28 29 30 31
    June
    MTWTFSS
    1 2 3 4 5 6 7
    8 9 10 11 12 13 14
    15 16 17 18 19 20 21
    22 23 24 25 26 27 28
    29 30
    July
    MTWTFSS
        1 2 3 4 5
    6 7 8 9 10 11 12
    13 14 15 16 17 18 19
    20 21 22 23 24 25 26
    27 28 29 30 31
    August
    MTWTFSS
              1 2
    3 4 5 6 7 8 9
    10 11 12 13 14 15 16
    17 18 19 20 21 22 23
    24 25 26 27 28 29 30
    31
    September
    MTWTFSS
      1 2 3 4 5 6
    7 8 9 10 11 12 13
    14 15 16 17 18 19 20
    21 22 23 24 25 26 27
    28 29 30
    October
    MTWTFSS
          1 2 3 4
    5 6 7 8 9 10 11
    12 13 14 15 16 17 18
    19 20 21 22 23 24 25
    26 27 28 29 30 31