Team:UNIPV-Pavia/Calendar/June/settimana4

From 2010.igem.org

(Difference between revisions)
(→June, 23rd)
(→June, 23rd)
Line 87: Line 87:
*I9: <partinfo>BBa_J23114</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)
*I10: <partinfo>BBa_J23116</partinfo> (S-P)+ I6 (X-P)
 +
 +
NanoDrop quantifications were not reliable, so every ligation was performed with 3ul of vector, 2ul of insert, 3ul of ddH20, 1ul of T4 ligase and 1ul of T4 ligase buffer.
These four promoters from Andersone Promoters Collection were choosed for their ''strength'', measured in Arbitrary Units:
These four promoters from Andersone Promoters Collection were choosed for their ''strength'', measured in Arbitrary Units:

Revision as of 08:56, 5 July 2010
Home
The Project
Materials & Methods
Parts
The Team
Notebook
Biological Safety
Pavia
Gallery
Sponsors

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)

NanoDrop quantifications were not reliable, so every ligation was performed with 3ul of vector, 2ul of insert, 3ul of ddH20, 1ul of T4 ligase and 1ul of T4 ligase buffer.

These four promoters from Andersone Promoters Collection were choosed for their strength, measured in Arbitrary Units:

Part RFP (a.u.)
<partinfo>BBa_J23118</partinfo>1429
<partinfo>BBa_J23110</partinfo> 844
<partinfo>BBa_J23114</partinfo> 256
<partinfo>BBa_J23116</partinfo> 396

Soon quantitative testes will be performed to quantify each promoter's strength in terms of RPU.

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"