Team:UNIPV-Pavia/Calendar/July/settimana3

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Revision as of 13:53, 26 July 2010

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

Trasformation of ligations in:

Ligation name	E. coli strain	Resistance
I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)	TOP10	Amp 100
I12= <partinfo>BBa_J23105</partinfo> (S-P) + I6 (X-P)	TOP10	Amp 100
I13= <partinfo>BBa_J23106</partinfo> (S-P) + I6 (X-P)	TOP10	Amp 100
I14= <partinfo>BBa_J23118</partinfo> (S-P) + I3 (X-P)	DH5alpha
I15= <partinfo>BBa_J23110</partinfo> (S-P) + I3 (X-P)	DH5alpha	Amp 100
I16= <partinfo>BBa_J23116</partinfo> (S-P) + I3 (X-P)	DH5alpha	Amp 100
I17= <partinfo>BBa_J23101</partinfo> (S-P) + I3 (X-P)	DH5alpha	Amp 100
I18= <partinfo>BBa_J23105</partinfo> (S-P) + I3 (X-P)	DH5alpha	Amp 100
I19= <partinfo>BBa_J23106</partinfo> (S-P) + I3 (X-P)	DH5alpha	Amp 100
I74C5= I7 (E-P) + 4C5 (E-P)	TOP10	Cm 12.5
I84C5= I8 (E-P) + 4C5 (E-P)	TOP10	Cm 12.5
I104C5= I10 (E-P) + 4C5 (E-P)	TOP10	Cm 12.5

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)

week 3

@@ Line 98: / Line 98: @@
 Trasformation of ligations in:
-<table width='80%' border='1'><tr>
+<table width='90%' border='1'>
+<tr><th>Ligation name</th><th>E. coli strain </th><th> Resistance </th></tr>
+<tr>
 <td>
 *I11= <partinfo>BBa_J23101</partinfo> (S-P) + I6 (X-P)
@@ Line 105: / Line 108: @@
 TOP10
 </td>
+<td>Amp 100</td>
 </tr><tr><td>
 *I12= <partinfo>BBa_J23105</partinfo> (S-P) + I6 (X-P)
@@ Line 111: / Line 115: @@
 TOP10
 </td>
+<td>Amp 100</td>
 </tr><tr><td>
 *I13= <partinfo>BBa_J23106</partinfo> (S-P) + I6 (X-P)
@@ Line 117: / Line 122: @@
 TOP10
 </td>
+<td>Amp 100</td>
 </tr><tr><td>
 *I14= <partinfo>BBa_J23118</partinfo> (S-P) + I3 (X-P)
@@ Line 129: / Line 135: @@
 DH5alpha
 </td>
+<td>Amp 100</td>
 </tr><tr><td>
 *I16= <partinfo>BBa_J23116</partinfo> (S-P) + I3 (X-P)
@@ Line 135: / Line 142: @@
 DH5alpha
 </td>
+<td>Amp 100</td>
 </tr><tr><td>
 *I17= <partinfo>BBa_J23101</partinfo> (S-P) + I3 (X-P)
@@ Line 141: / Line 149: @@
 DH5alpha
 </td>
+<td>Amp 100</td>
 </tr><tr><td>
 *I18= <partinfo>BBa_J23105</partinfo> (S-P) + I3 (X-P)
@@ Line 147: / Line 156: @@
 DH5alpha
 </td>
+<td>Amp 100</td>
 </tr><tr><td>
 *I19= <partinfo>BBa_J23106</partinfo> (S-P) + I3 (X-P)
@@ Line 153: / Line 163: @@
 DH5alpha
 </td>
+<td>Amp 100</td>
 </tr><tr><td>
 *I74C5= I7 (E-P) + 4C5 (E-P)
@@ Line 159: / Line 170: @@
 TOP10
 </td>
+<td>Cm 12.5</td>
 </tr><tr><td>
 *I84C5= I8 (E-P) + 4C5 (E-P)
@@ Line 165: / Line 177: @@
 TOP10
 </td>
+<td>Cm 12.5</td>
 </tr><tr><td>
 *I104C5= I10 (E-P) + 4C5 (E-P)
@@ Line 171: / Line 184: @@
 TOP10
 </td>
+<td>Cm 12.5</td>
 </tr>
 </table>