Team:UNIPV-Pavia/Calendar/June/settimana4

From 2010.igem.org

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==June, 23rd==
==June, 23rd==
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Cultures incubated ON were all grown. Plasmids were extracted with MiniPrep.
[[Image:Unipv_digestion_J231xx_I6.jpg|300px|thumb|center|J231xx and I6 digestion]]
[[Image:Unipv_digestion_J231xx_I6.jpg|300px|thumb|center|J231xx and I6 digestion]]
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 +
After MiniPrep, purified DNA was quantified with NanoDrop.
 +
 +
{| border="1"
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|  I6  ||  193 ng/ul 
 +
|-
 +
|  <partinfo>BBa_J23118</partinfo>    ||  107,9 ng/ul     
 +
|-
 +
|  <partinfo>BBa_J23110</partinfo>    ||  83 ng/ul   
 +
|-
 +
|  <partinfo>BBa_J23114</partinfo>    ||  89,8 ng/ul     
 +
|-
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|  <partinfo>BBa_J23116</partinfo>    ||  82,7 ng/ul   
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|}
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</td>
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</tr>
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</table>
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Digestion of:
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 +
{| border="1"
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|  ''Culture''    ||  ''Kind''  ||  ''Final reaction volume (ul) '' ||  ''DNA (ul)''  ||  ''H20 (ul)''  ||  ''Enzyme 1''  ||  ''Enzyme 2'' ||  ''Buffer H''
 +
|-
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|  I6  ||  Insert  || 25 ||  9,3  ||  11,2  ||      1 XbaI  || 1 PstI  || 2,5
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|-
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|  I6-bis  ||  Insert  ||    25 ||9,3  ||  11,2  ||      1 XbaI  || 1 PstI  || 2,5
 +
|-
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|  <partinfo>BBa_J23118</partinfo>    ||  Vector  ||  25 || 9,3  ||  11,2  ||      1 SpeI  || 1 PstI  || 2,5     
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|-
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|  <partinfo>BBa_J23110</partinfo>    ||  Vector  ||    25 ||12  ||  8,5  ||      1 SpeI  || 1 PstI  || 2,5     
 +
|-
 +
|  <partinfo>BBa_J23114</partinfo>    ||  Vector  ||  25 ||  11,2  ||  9,3  ||      1 SpeI  || 1 PstI  || 2,5 
 +
|-
 +
|  <partinfo>BBa_J23116</partinfo>    ||  Vector  ||  25 || 12,1  ||  8,4  ||      1 SpeI  || 1 PstI  || 2,5 
 +
|}
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 +
Digestions were incubated at 37°C for 3 hours, gel run and gel-extracted.
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 +
Ligations were performed ON at 16°C:
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 +
*I7: <partinfo>BBa_J23118</partinfo> (S-P)+ I6 (X-P)
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*I8: <partinfo>BBa_J23110</partinfo> (S-P)+ I6 (X-P)
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*I9: <partinfo>BBa_J23114</partinfo> (S-P)+ I6 (X-P)
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*I10: <partinfo>BBa_J23116</partinfo> (S-P)+ I6 (X-P)
==June, 24th==
==June, 24th==

Revision as of 08:26, 5 July 2010
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JUNE: WEEK 4


June, 21st

Planning of the activity of the week. Freezer cleaning: for each ligation, we choosed the correct clone and stored it in the iGEM 2010 ligations box. All these cloned were gel screened and sequenced and re correct! The colonies we choosed are:

colony choosed ligation name
I0-2I0
I1-2I1
I2-1I2
I3-1I3
I4-2I4
I5-1I5
I6-2I6

Other colonies resulted psitives at the screening but were NOT sequenced and are stored in the iGEM 2010 cemetery box. These clones are: I1-1, I2-2, I4-1 and I6-3.

June, 22nd

Inoculum of I6, <partinfo>BBa_J23118</partinfo>, <partinfo>BBa_J23110</partinfo>, <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_J23116</partinfo> from glycerol stock in 5ml LB+Amp. Cultures were grown ON at 37°C 220rpm.

June, 23rd

Cultures incubated ON were all grown. Plasmids were extracted with MiniPrep.


J231xx and I6 digestion

After MiniPrep, purified DNA was quantified with NanoDrop.

I6 193 ng/ul
<partinfo>BBa_J23118</partinfo> 107,9 ng/ul
<partinfo>BBa_J23110</partinfo> 83 ng/ul
<partinfo>BBa_J23114</partinfo> 89,8 ng/ul
<partinfo>BBa_J23116</partinfo> 82,7 ng/ul

Digestion of:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 Enzyme 2 Buffer H
I6 Insert 25 9,3 11,2 1 XbaI 1 PstI 2,5
I6-bis Insert 25 9,3 11,2 1 XbaI 1 PstI 2,5
<partinfo>BBa_J23118</partinfo> Vector 25 9,3 11,2 1 SpeI 1 PstI 2,5
<partinfo>BBa_J23110</partinfo> Vector 25 12 8,5 1 SpeI 1 PstI 2,5
<partinfo>BBa_J23114</partinfo> Vector 25 11,2 9,3 1 SpeI 1 PstI 2,5
<partinfo>BBa_J23116</partinfo> Vector 25 12,1 8,4 1 SpeI 1 PstI 2,5

Digestions were incubated at 37°C for 3 hours, gel run and gel-extracted.

Ligations were performed ON at 16°C:

  • I7: <partinfo>BBa_J23118</partinfo> (S-P)+ I6 (X-P)
  • I8: <partinfo>BBa_J23110</partinfo> (S-P)+ I6 (X-P)
  • I9: <partinfo>BBa_J23114</partinfo> (S-P)+ I6 (X-P)
  • I10: <partinfo>BBa_J23116</partinfo> (S-P)+ I6 (X-P)

June, 24th

June, 25th

Screening of I7, I8, I9, I10

June

week 2

week 3

week 4


</td>

Retrieved from "http://2010.igem.org/Team:UNIPV-Pavia/Calendar/June/settimana4"