Team:UNIPV-Pavia/Calendar/July/settimana3

From 2010.igem.org

(Difference between revisions)
(→July, 12th)
(→July, 13th)
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==July, 13th==
==July, 13th==
 +
LB+Amp was prepared and phasins sample were sent to be sequenced.
 +
 +
For cultures grown OverNight at 37°C, 220 rpm were MiniPrep was performed:
 +
 +
{| border='1' align='center'
 +
|| <partinfo>BBa_J23101</partinfo> || XXX ng/ul
 +
|-
 +
|| <partinfo>BBa_J23105</partinfo> || XXX ng/ul
 +
|-
 +
|| <partinfo>BBa_J23106</partinfo> || XXX ng/ul
 +
|}
 +
 +
Other plasmids were retrieved from our freezer:
 +
 +
*<partinfo>BBa_J23118</partinfo> (already digested SpeI-PstI)
 +
*<partinfo>BBa_J23110</partinfo> (already digested SpeI-PstI)
 +
*<partinfo>BBa_J23116</partinfo> (already digested SpeI-PstI)
 +
*I6-2 (already digested XbaI-PstI)
 +
*4C5 (MiniPrep performed, it will be digested EcoRI-PstI)
 +
*I7-3 (MiniPrep performed, it will be digested EcoRI-PstI)
 +
*I8-5 (MiniPrep performed, it will be digested EcoRI-PstI)
 +
*I10-1 (MiniPrep performed, it will be digested EcoRI-PstI)
 +
*I3-1 (MiniPrep performed, it will be digested XbaI-PstI)
 +
 +
 +
Digestion of:
 +
 +
{| border="1" align='center'
 +
|  ''Culture''    ||  ''Kind''  ||  ''Final reaction volume (ul) '' ||  ''DNA (ul)''  ||  ''H20 (ul)''  ||  ''Enzyme 1''  ||  ''Enzyme 2'' ||  ''Buffer H''
 +
|-
 +
|  4C5 (x2)  ||  Vector || 25 ||  3,6  ||  16,9  ||      1 EcoRI  || 1 PstI  || 2,5
 +
|-
 +
|  I7-3  ||  Insert || 25 ||  14  ||  6,5  ||      1 EcoRI  || 1 PstI  || 2,5
 +
|-
 +
|  I8-5  ||  Insert || 25 ||  12,7  ||  7,8  ||      1 EcoRI  || 1 PstI  || 2,5
 +
|-
 +
|  I10-1  ||  Insert || 25 ||  15,3  ||  5,2  ||      1 EcoRI  || 1 PstI  || 2,5
 +
|-
 +
|  I3-1 (x2)  ||  Vector || 25 ||  5,8  ||  14,7  ||      1 XbaI  || 1 PstI  || 2,5
 +
|-
 +
|  <partinfo>BBa_J23105</partinfo>  ||  Insert || 25 ||  15,9  ||  4,6  ||      1SpeI  || 1 PstI  || 2,5
 +
|-
 +
|  <partinfo>BBa_J23106</partinfo>  ||  Insert || 25 ||  13  ||  7,5  ||      1 SpeI  || 1 PstI  || 2,5
 +
|-
 +
|  <partinfo>BBa_J23101</partinfo>  ||  Insert || 25 ||  10,4  ||  10,1  ||      1 SpeI  || 1 PstI  || 2,5
 +
|}
 +
 +
Digestions were incubated at 37°C for 3hours, then gel run/cut.
 +
 +
Gel was prepared for electrophoresis: 150ml TBE + 1,5 g Agarose + 3ul EtBr.
 +
 +
Ligations were all performed 1:5 (1ul vector + 5ul insert):
 +
 +
*I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)
 +
*I12= <partinfo>BBa_J23105</partinfo> (S-P) + I6 (X-P)
 +
*I13= <partinfo>BBa_J23106</partinfo> (S-P) + I6 (X-P)
 +
*I14= <partinfo>BBa_J23118</partinfo> (S-P) + I3 (X-P)
 +
*I15= <partinfo>BBa_J23110</partinfo> (S-P) + I3 (X-P)
 +
*I16= <partinfo>BBa_J23116</partinfo> (S-P) + I3 (X-P)
 +
*I17= <partinfo>BBa_J23101</partinfo> (S-P) + I3 (X-P)
 +
*I18= <partinfo>BBa_J23105</partinfo> (S-P) + I3 (X-P)
 +
*I19= <partinfo>BBa_J23106</partinfo> (S-P) + I3 (X-P)
 +
*I74C5= I7 (E-P) + 4C5 (E-P)
 +
*I84C5= I8 (E-P) + 4C5 (E-P)
 +
*I104C5= I10 (E-P) + 4C5 (E-P)
==July, 14th==
==July, 14th==

Revision as of 13:45, 26 July 2010
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JULY: WEEK 3


July, 12th

Phasins PhaP1 and PhaP2 were sequenced, but none of them has a clear chromatogram, so we decided to sequence the PCR results.

Gel run/cut of phasins amplified via PCR

Phasins were gel run/cut and the samples were prepared to be sequenced.

Inoculum of

  • <partinfo>BBa_J23101</partinfo>
  • <partinfo>BBa_J23105</partinfo>
  • <partinfo>BBa_J23106</partinfo>

for tomorrow ligations.

July, 13th

LB+Amp was prepared and phasins sample were sent to be sequenced.

For cultures grown OverNight at 37°C, 220 rpm were MiniPrep was performed:

<partinfo>BBa_J23101</partinfo> XXX ng/ul
<partinfo>BBa_J23105</partinfo> XXX ng/ul
<partinfo>BBa_J23106</partinfo> XXX ng/ul

Other plasmids were retrieved from our freezer:

  • <partinfo>BBa_J23118</partinfo> (already digested SpeI-PstI)
  • <partinfo>BBa_J23110</partinfo> (already digested SpeI-PstI)
  • <partinfo>BBa_J23116</partinfo> (already digested SpeI-PstI)
  • I6-2 (already digested XbaI-PstI)
  • 4C5 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I7-3 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I8-5 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I10-1 (MiniPrep performed, it will be digested EcoRI-PstI)
  • I3-1 (MiniPrep performed, it will be digested XbaI-PstI)


Digestion of:

Culture Kind Final reaction volume (ul) DNA (ul) H20 (ul) Enzyme 1 Enzyme 2 Buffer H
4C5 (x2) Vector 25 3,6 16,9 1 EcoRI 1 PstI 2,5
I7-3 Insert 25 14 6,5 1 EcoRI 1 PstI 2,5
I8-5 Insert 25 12,7 7,8 1 EcoRI 1 PstI 2,5
I10-1 Insert 25 15,3 5,2 1 EcoRI 1 PstI 2,5
I3-1 (x2) Vector 25 5,8 14,7 1 XbaI 1 PstI 2,5
<partinfo>BBa_J23105</partinfo> Insert 25 15,9 4,6 1SpeI 1 PstI 2,5
<partinfo>BBa_J23106</partinfo> Insert 25 13 7,5 1 SpeI 1 PstI 2,5
<partinfo>BBa_J23101</partinfo> Insert 25 10,4 10,1 1 SpeI 1 PstI 2,5

Digestions were incubated at 37°C for 3hours, then gel run/cut.

Gel was prepared for electrophoresis: 150ml TBE + 1,5 g Agarose + 3ul EtBr.

Ligations were all performed 1:5 (1ul vector + 5ul insert):

  • I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)
  • I12= <partinfo>BBa_J23105</partinfo> (S-P) + I6 (X-P)
  • I13= <partinfo>BBa_J23106</partinfo> (S-P) + I6 (X-P)
  • I14= <partinfo>BBa_J23118</partinfo> (S-P) + I3 (X-P)
  • I15= <partinfo>BBa_J23110</partinfo> (S-P) + I3 (X-P)
  • I16= <partinfo>BBa_J23116</partinfo> (S-P) + I3 (X-P)
  • I17= <partinfo>BBa_J23101</partinfo> (S-P) + I3 (X-P)
  • I18= <partinfo>BBa_J23105</partinfo> (S-P) + I3 (X-P)
  • I19= <partinfo>BBa_J23106</partinfo> (S-P) + I3 (X-P)
  • I74C5= I7 (E-P) + 4C5 (E-P)
  • I84C5= I8 (E-P) + 4C5 (E-P)
  • I104C5= I10 (E-P) + 4C5 (E-P)

July, 14th

Quality control for HELPER and CRIM plasmids

July, 15th

Quality control for pBHR68 and CRIM plasmids

July, 16th

Screening ligations I11-I16
Screening ligations I17-I19, I7/I8/I10-4C5


Since it's impossible to isolate the CRIM plasmid <partinfo>BBa_J72007</partinfo> we created a pseudo CRIM plasmid to check that not pir strains aren't able to propagate it. We called it "THE RING" (RING) and we built it from our I5 (Cm resistance - <partinfo>BBa_P1004</partinfo> - + TT - <partinfo>BBa_B0015</partinfo> - + R6K ori - <partinfo>BBa_J61001</partinfo>) in <partinfo>pSB1A2</partinfo>:

  • Digestion of I5 XbaI-PstI
  • Gel extraction
  • Quantification with Nanodrop (15ng/ul)
  • Ligation of X and P sites each other (used 2ul of DNA)


Calendar

July

week 1

week 2

week 3

week 4

week 5

Retrieved from "http://2010.igem.org/Team:UNIPV-Pavia/Calendar/July/settimana3"