Team:UNIPV-Pavia/Calendar/July/settimana4

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Revision as of 00:51, 19 July 2011

UNIPV TEAM 2011

March
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April
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May
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30 31

June
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13 14 15 16 17 18 19
20 21 22 23 24 25 26
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July
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18 19 20 21 22 23 24
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August
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September
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October
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31

JULY: WEEK 3

July, 11th

Digestions of previously purified plasmids were performed for ligations:

CAMBIARE OGGETTI TABELLA

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
<partinfo>BBa_C0060</partinfo> Insert 16.5 4 1 EcoRI 1 SpeI 2.5 25
<partinfo>BBa_C0061</partinfo> Insert 13.2 7.3 1 XbaI 1 PstI 2.5 25
<partinfo>BBa_K081022</partinfo> Insert 15.7 4.8 1 EcoRI 1 PstI 2.5 25
<partinfo>BBa_B0030</partinfo> Vector 13.2 7.3 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_B0031</partinfo> Vector 12.4 8.1 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_B0032</partinfo> Vector 9.5 11 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_B0015</partinfo> Vector 7.9 12.6 1 EcoRI 1 XbaI 2.5 25
<partinfo>pSB4C5</partinfo> Vector 3 17.5 1 EcoRI 1 PstI 2.5 25
<partinfo>BBa_I13501</partinfo> Insert 7.2 13.3 1 XbaI 1 PstI 2.5 25

Reactions were incubated at 37°C for three hours while two medium-size agarose gels were prepared according to protocols.

In the afternoon gel electrophoresis was performed.

2 righe sopra inserire l' immagine

As shown, in figure all clones were positive (apart from <partinfo>BBa_I13501</partinfo> run where we inexplicably didn't see any band), so we cut and purified the bands of interest. Cells harbouring <partinfo>BBa_I13501</partinfo> were again inoculated in 5 ml LB + Amp.

After gel extraction, cut DNA was quantified:

Plasmid DNA (ng/μl)
<partinfo>BBa_C0060</partinfo> (E-S) 3.9
<partinfo>BBa_C0061</partinfo> (X-P) 4.1
<partinfo>BBa_K081022</partinfo> (E-P) 4.4
<partinfo>BBa_B0030</partinfo> (S-P) 10.3
<partinfo>BBa_B0031</partinfo> (S-P) 11.8
<partinfo>BBa_B0032</partinfo> (S-P) 10.1
<partinfo>BBa_B0015</partinfo> (E-X) 12
<partinfo>pSB4C5</partinfo> (E-P) 10.7

Then ligations were performed in a final volume of 10 μl:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
E1 <partinfo>BBa_B0015</partinfo> (E-X) 1.5 <partinfo>BBa_C0060</partinfo> (E-S) 6.5 1 1
E2 <partinfo>BBa_B0030</partinfo> (S-P) 1.5 <partinfo>BBa_C0061</partinfo> (X-P) 6.5 1 1
E3 <partinfo>BBa_B0031</partinfo> (S-P) 1.5 <partinfo>BBa_C0061</partinfo> (X-P) 6.5 1 1
E4 <partinfo>BBa_B0032</partinfo> (S-P) 1.5 <partinfo>BBa_C0061</partinfo> (X-P) 6.5 1 1
E8 <partinfo>pSB4C5</partinfo> (E-P) 1.5 <partinfo>BBa_K081022</partinfo> (E-P) 6.5 1 1

Ligations were incubated ON at 16°C.

July, 19th