Difference between revisions of "Team:Marburg/Labbook/CDI"

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 +
<h1>15/05/21</h1>
  
 +
<h2>PCR</h2>
  
 +
<p>
 +
Primers ICD014 and ICD015 arrived! These will be used to amplify the Pick up backbone with overlaps for the full-length Cdi operon. PCR should yield a 3391 bp fragment.
 +
<br>
 +
<table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
 +
  <tr style="background-color:#f66300;">
 +
        <td>PCR </td>
 +
        <td>PCR Mix 1 </td>
 +
     
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>GXL buffer</td>
 +
        <td class='col'>10 uL </td>
 +
       
 +
       
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>dNTPs (from GXL kit</td>
 +
        <td class='col'>4 uL</td>
 +
       
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>GXL polymerase</td>
 +
        <td class='col'>2uL</td>
 +
       
 +
    </tr>
 +
  <tr>
 +
        <td class='col'>ICD0014 (10 uM)</td>
 +
          <td class='col'>1 uL</td>
 +
           
 +
    </tr>
 +
  <tr>
 +
        <td class='col'>ICD0015 (10 uM)</td>
 +
        <td class='col'>1uL</td>
 +
     
 +
     
 +
    </tr>
 +
  <tr>
 +
        <td class='col'>1:10 diluted plasmid X</td>
 +
        <td class='col'>1 uL</td>
 +
       
 +
    </tr>
 +
  <tr>
 +
        <td class='col'>ddH2O</td>
 +
        <td class='col'>31 uL</td>
 +
       
 +
    </tr>
  
 +
</table>
  
 +
</PCR program>
  
 +
<table id="layouttable" style="border:2px;border-style:solid;border-color:#F0F0F0;">
 +
  <tr style="background-color:#f66300;">
 +
        <td>Program </td>
 +
        <td> time </td>
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>Start cycle (30x)</td>
 +
        <td class='col'> </td>
 +
       
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>98 °C </td>
 +
        <td class='col'>10 s </td>
 +
       
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>60 °C</td>
 +
        <td class='col'>15 s</td>
 +
    </tr>
 +
    <tr>
 +
        <td class='col'>68 °C</td>
 +
        <td class='col'>35 s</td>
 +
    </tr>
 +
  <tr>
 +
        <td class='col'>close cycle</td>
 +
          <td class='col'> </td>
 +
     
 +
    </tr>
 +
  <tr>
 +
        <td class='col'> 8 °C</td>
 +
        <td class='col'>store</td>
 +
     
 +
    </tr>
 +
 +
</table>
  
 
<img src="https://static.igem.org/mediawiki/2015/f/f3/MR_pic_NUTRInitybig.png" class="imgcenter;" style="width:200px;">
 
<img src="https://static.igem.org/mediawiki/2015/f/f3/MR_pic_NUTRInitybig.png" class="imgcenter;" style="width:200px;">

Revision as of 20:27, 12 September 2015

15/05/18

PCR

PCR PCR Mix 1 PCR Mix 2
GXL buffer 10 uL 10 uL
dNTPs (from GXL kit 4 uL 4 uL
GXL polymerase 2uL 2uL
ICD001 (10 uM) 0.5uL 1uL
ICD007/ICD008 (10 uM) 0.5uL 1uL
EC93 genomic DNA (15 ng/uL) 0.5 uL 1 uL
ddH2O 32.5uL 29.5uL
Program time
Start cycle (30x)
98 °C 10 s
60 °C 15 s
68 °C 2 min
close cycle
8 °C store
ILS
Figure 1: Ladder, PCR 1, PCR 2

Ran of analytical gel. No bands were observed.

15/05/19

PCR

ILS
Figure 1: Ladder, PCR 1, PCR 2

Bands visible! Lanes 1 and 2 right of the ladder should have a 10506 bp band. Lanes 3 and 4 right of the ladder should have a 11414 bp band (gel description should read EC93 instead of EC90).
PCR cleanup (according to Thermo Scientific protocol), elution in 21 ul.
Concentrations according to Nanodrop: EC93 w/ 001+007: 92 ng/ul; EC93 w/ 001+008: 57 ng/ul

15/05/21

PCR

Primers ICD014 and ICD015 arrived! These will be used to amplify the Pick up backbone with overlaps for the full-length Cdi operon. PCR should yield a 3391 bp fragment.

PCR PCR Mix 1
GXL buffer 10 uL
dNTPs (from GXL kit 4 uL
GXL polymerase 2uL
ICD0014 (10 uM) 1 uL
ICD0015 (10 uM) 1uL
1:10 diluted plasmid X 1 uL
ddH2O 31 uL
Program time
Start cycle (30x)
98 °C 10 s
60 °C 15 s
68 °C 35 s
close cycle
8 °C store

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