Team:UNIPV-Pavia/Calendar/July/settimana3

From 2011.igem.org

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Revision as of 00:13, 20 July 2011

UNIPV TEAM 2011

March
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    

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
          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

JULY: WEEK 3

July, 11th

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
E1-2 Insert 18 2.5 1 XbaI 1 PstI 2.5 25
E2-2 Insert 18 2.5 1 XbaI 1 PstI 2.5 25
E3-1 Insert 18 2.5 1 XbaI 1 PstI 2.5 25
E4-2 Insert< 18 2.5 1 XbaI 1 PstI 2.5 25
E5-2 Insert 18 2.5 1 XbaI 1 PstI 2.5 25
E6-1 Insert 11 9.5 1 XbaI 1 PstI 2.5 25
E7-2 Insert 18 2.5 1 XbaI 1 PstI 2.5 25
E8-3 Vector 20.5 0 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_R0040</partinfo> Vector 14.5 6 1 SpeI 1 PstI 2.5 25
<partinfo>BBa_C0261</partinfo> Insert< 18 2.5 1 XbaI 1 PstI 2.5 25
<partinfo>BBa_I13507</partinfo> Insert 10 10.5 1 XbaI 1 PstI 2.5 25
<partinfo>BBa_B0034</partinfo> Vector 10 10.5 1 SpeI 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. 

                
 link alle 2 img dei gel con ling wiki

As shown, in figure all clones were positive, so we cut and purified the bands of interest.

After gel extraction, cut DNA was quantified:

Plasmid DNA (ng/μl)
E1 (X-P) 4.2
E2 (X-P) 3.8
E3 (X-P) 4.1
E4 (X-P) 4.2
E5 (X-P) 5.6
E6 (X-P) 7.8
E7 (X-P) 6.4
E8 (S-P) 3.4
<partinfo>BBa_C0261</partinfo> (X-P) 4.8
<partinfo>BBa_R0040</partinfo> (S-P) 7.9
<partinfo>BBa_B0034</partinfo> (S-P) 11.0
<partinfo>BBa_I13507</partinfo> (X-P) 3.9


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)
E9 <partinfo>BBa_B0030</partinfo> (S-P) 1 E1 (X-P) 7 1 1
E10 <partinfo>BBa_B0031</partinfo> (S-P) 1 E1 (X-P) 7 1 1
E11 <partinfo>BBa_B0032</partinfo> (S-P) 1 E1 (X-P) 7 1 1
E12 <partinfo>BBa_B0034</partinfo> (S-P) 1 E1 (X-P) 7 1 1
E13 <partinfo>BBa_R0040</partinfo> (S-P) 1.5 E2 (X-P) 6.5 1 1
E14 <partinfo>BBa_R0040</partinfo> (S-P) 1.5 E3 (X-P) 6.5 1 1
E15 <partinfo>BBa_R0040</partinfo> (S-P) 1.5 E4 (X-P) 6.5 1 1
E16 <partinfo>BBa_R0040</partinfo> (S-P) 2 <partinfo>BBa_C0261</partinfo> (X-P) 6 1 1
E17 E8 (S-P) 4.5 E5 (X-P) 3.5 1 1
E18 E8 (S-P) 5 E6 (X-P) 3 1 1
E19 E8 (S-P) 5 E7 (X-P) 3 1 1
E20 E8 (S-P) 5 <partinfo>BBa_I13507</partinfo> (X-P) 3 1 1
E21 <partinfo>BBa_R0040</partinfo> (S-P) 2 E5 (X-P) 6 1 1
E22 <partinfo>BBa_R0040</partinfo> (S-P) 2.5 E6 (X-P) 5.5 1 1
E23 <partinfo>BBa_R0040</partinfo> (S-P) 2 E7 (X-P) 6 1 1

Ligations were incubated ON at 16°C.

July, 12th

E9, E10, E11, E12, E13, E14, E15, E16, E17, E18, 19, E20, E21, E22, E23, and E8 were transformed in 100 μl of TOP10 competent cells according to protocols. Plates were incubated ON at 37°C.

July, 13th

All plates showed a lot of colonies, except for E13, E16 (one colony),while for E14, E15 no colony was observed. Because of RFP, E21 was red, E22 and E23 a little bit less. Two colonies for E9, E10, E11, E12, E17, E18, E19, E20, E21, E22, E23 plates were picked. 500 ml of LB with Ampicillin were prepared. ??????????????appunti nico?????????????????????-->stock

July, 14th

A glycerol stock for E21-2, E23-2. E11-1 was prepared. Cultures were saturated; plasmid purification was carried out:

Plasmid DNA (ng/μl)
E9-1 114
E10-1 72
E11-1 78.5
E12-1 1151.7
E13-1 133
E16-1 134.4
E17-1 27.8
E18-1 25.5

For E9-1, E9-2, E10-1, E10-2, E11-1, E11-2, E12-1, E12-2, E17-1, E18-2, E19-1, E19-2, E21-1, E20-2, E21-1, E21-2, E22-1, E22-2, E23-1, E23-2 PCR was performed. ??????????????????????????????????primer? da scrivere??????????????????????????????????????? <partinfo>BBa_B0021</partinfo>were transformed to test the competence?.

In order to screen the DNA, digestions for each ligation was performed for screening.

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
Each ligation 3 18.5 0.5 EcoRI 0.5 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. 

                
 link alle 2 img dei gel con ling wiki

As shown in figure, all clones were positive, so we cut and purified the bands of interest, except for E9-1,E17-2, E18-1, E18-2, E19-1 which didn' t show any DNA band. FINIRE QUESTA GIORNATA

After gel extraction, cut DNA was quantified:



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University of Pavia

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