Team:UNIPV-Pavia/Calendar/July/settimana4

From 2011.igem.org

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Revision as of 10:01, 21 July 2011

UNIPV TEAM 2011

March
M T W T F S S
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7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 31    

April
M T W T F S S
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30

May
M T W T F S S
            1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31

June
M T W T F S S
    1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30

July
M T W T F S S
        1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31

August
M T W T F S S
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31

September
M T W T F S S
      1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30

October
M T W T F S S
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3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31

JULY: WEEK 4

July, 18th

Digestions of previously purified plasmids were performed for ligations:

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
E9-2 Insert 11.5 9 1 Xbal 1 Pstl 2.5 25
E10-1 Insert 10 10.5 1 Xbal 1 Pstl 2.5 25
E11-1 Insert 4 16.5 1 Xbal 1 Pstl 2.5 25
E12-1 Insert 10 10.5 1 Xbal 1 PstI 2.5 25
E13-1 ??Insert?? 6 14.5 1 EcoRl 1 Pstl 2.5 25
E16-1 ??Insert?? 4 16.5 1 EcoRl 1 Pstl 2.5 25
E21-1 ??Insert?? 7.5 13 1 EcoRl 1 Xbal 2.5 25
E22-2 ??Insert?? 10 10.5 1 EcoRl 1 Pstl 2.5 25
<partinfo>BBa_I13521</partinfo> Insert 12.5 8 1 EcoRl 1 Pstl 2.5 25
<partinfo>BBa_R0040</partinfo>-<partinfo>BBa_J61002</partinfo> Vector 20.5 0 1 EcoRl 1 Pstl 2.5 25

Reactions were incubated at 37°C for three hours; meanwhile two medium-size agarose gels were prepared according to protocols. Moreover BBa_I13521 plasmid purification and quantification were carried out:

In the afternoon gel electrophoresis was performed.

Plasmid DNA (ng/μl)
<partinfo>BBa_I13521</partinfo> 37.3

2 righe sopra inserire l' immagine

As shown in figure, all clones were positive (apart from E13 run where we inexplicably didn't see any band), so we cut and purified the bands of interest. Cells harbouring E13 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