Team:UNIPV-Pavia/Calendar/July/settimana5

From 2011.igem.org

(Difference between revisions)
Line 41: Line 41:
       <td>13</td>
       <td>13</td>
       <td>7.5</td>
       <td>7.5</td>
-
       <td>1 Xbal</td>
+
       <td>1 XbaI</td>
-
       <td>1 Pstl</td>
+
       <td>1 PstI</td>
       <td>2.5</td>
       <td>2.5</td>
       <td>25</td>
       <td>25</td>
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       <td>13.5</td>
       <td>13.5</td>
       <td>7</td>
       <td>7</td>
-
       <td>1 Xbal</td>
+
       <td>1 XbaI</td>
-
       <td>1 Pstl</td>
+
       <td>1 PstI</td>
       <td>2.5</td>
       <td>2.5</td>
       <td>25</td>
       <td>25</td>
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       <td>14.5</td>
       <td>14.5</td>
       <td>6</td>
       <td>6</td>
-
       <td>1 Xbal</td>
+
       <td>1 XbaI</td>
-
       <td>1 Pstl</td>
+
       <td>1 PstI</td>
       <td>2.5</td>
       <td>2.5</td>
       <td>25</td>
       <td>25</td>
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       <td>12.5</td>
       <td>12.5</td>
       <td>8</td>
       <td>8</td>
-
       <td>1 Xbal</td>
+
       <td>1 XbaI</td>
       <td>1 PstI</td>
       <td>1 PstI</td>
       <td>2.5</td>
       <td>2.5</td>

Revision as of 21:08, 16 August 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 5

July, 25th

Red colonies were observed from E36 plate, suggesting that the transformation was successful. A colony was picked form the plate.

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)
E24-2 Insert 13 7.5 1 XbaI 1 PstI 2.5 25
E25-1 Insert 13.5 7 1 XbaI 1 PstI 2.5 25
E26-2 Insert 14.5 6 1 XbaI 1 PstI 2.5 25
E27-2 Insert 12.5 8 1 XbaI 1 PstI 2.5 25

Reactions were incubated at 37°C for three hours while a small size agarose gel was prepared according to protocols.
In the afternoon gel electrophoresis was performed:

Small size gel

After gel extraction, digested DNA was quantified:

Part DNA (ng/μl)
E24-2 (E-P) 7.4
E25-1 (E-P) 8.4
E26-2 (E-P) 3.2
E27-2 (E-P) 6.3

Ligations were performed:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
E37 pSB4C5 (E-P) 2 E24-2 (E-P) 6 1 1
E38 pSB4C5 (E-P) 2.5 E25-1 (E-P) 5.5 1 1
E39 pSB4C5 (E-P) 1 E26-2 (E-P) 7 1 1
E40 pSB4C5 (E-P) 2 E27-2 (E-P) 6 1 1

Inoculum of E17-2, E18-2, E19-2 or E20-2 to amplify DNA and transfer it into MGZ1; refill for E36 liquid culture.
Glycerol stock fpr E36 was prepared
250 ml of LB + Cm 12.5 were prepared.

July, 26th

Plasmids containing E17-2, E18-2, E19-2, E20-2 and E36 were extracted with MiniPrep kit from ON saturated cultures; purified DNA was quantified with NanoDrop Spectrophotometer:

Plasmid DNA (ng/μl)
E17-2 20.3
E18-2 18.6
E19-2 17.3
E20-2 13.2
E36 17.8

Digestion of E36 was performed for ligations:

Plasmid Kind DNA (μl) H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl) Final Volume (μl)
E36 Vector 20.5 0 1 Spel 1 Pstl 2.5 25

The sample was incubated at 37°C for three hours while a small-size agarose gel was prepared; in the afteroon gel electrophoresis was carried out:

Small size gel

After gel extraction, digested DNA was quantified:

Part DNA (ng/μl)
E36 (S-P) 3.5

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)
E41 E36 (S-P) 4 E3-1 (X-P) 4 1 1
E42 E36 (S-P) 3.5 E4-2 (X-P) 4.5 1 1

Ligations were incubated at 16°C ON.

July, 27th

E37, E38, E39, E40, E41, E42, E17, E18, E19 and E20 were transformed in 100 μl of MGZ1 competent cells according to protocols. Moreover 4ng of purified BBa_B0032 DNA were transformed in MGZ1 in order to test the transformation efficiency. Plates were incubated ON at 37°C.
BMR Genomics sent results of parts sequencing; E17-2, E18-2, E31-1, E32-2, E33-2, E34-1, E35-2 were OK while E28-1 sequence was wrong.

July, 28th

Colonies grew in every plate. In fact E17-2, E18-2, E19-2 and E20-2, E37, E38, E39 and E40 showed grown colonies. We picked two colonies for each plate and inoculate them in 5 ml of LB + Cm 12.5. As regards to E41 and E42 parts, we resolved to let them still grow; in the late morning we picked and inoculate two colonies for both plates.
Plate with BBa_B0032 showed 9 colonies; the corresponding efficiency results in 2250 colonies/μg of DNA (very low, we need to prepare new competent MGZ1).

July, 29th

Plasmid purification was carried out on over-night grown cultures (E41-1 did not grow!):

Plasmid DNA (ng/μl)
E37-1 16.6
E37-2 16.9
E38-1 28.4
E38-2 15.0
E39-1 15.0
E39-2 14.5
E40-1 17.1
E40-2 38.9
E41-2 13.7
E42-1 22.7
E42-2 28

A 12x mix was prepared for screening digestions:

H2O (μl) Enzyme 1 (μl) Enzyme 2 (μl) Buffer H (μl)
126 12 EcoRI 12 PstI 30

For each digestion 10 μl of purified DNA were used. Meanwhile a medium size gel was prepared.
In the afternoon gel electrophoresis was carried out:

Medium size gel

All clones were positive except for E42-2 and E41-2.
Glycerol stocks were prepared for E37-2, E38-1, E39-1, E40-2 and E42-1.
Ligations of consitutive promoter BBa_J23101 with different RBS in pSB4C5 were performed:

Ligation Name Vector Vector volume (μl) Insert Insert volume (μl) Buffer (μl) T4 Ligase (μl)
BBa_J23101-31 BBa_J23101 in pSB4C5 (S-P) 4 E6 (X-P) 4 1 1
BBa_J23101-E5 BBa_J23101 in pSB4C5 (S-P) 4 E5 (X-P) 4 1 1
BBa_J23101-E7 BBa_J23101 in pSB4C5 (S-P) 4 E7 (X-P) 4 1 1

July, 30th

E28 and E41 were again transformed in 200 μl of MGZ1 competent cells.
J101-E5, J101-31 and J101-E7 ligations were transformed in 100 μl of TOP10 competent cells.

July, 31st

All plates showed colonies. One colony was picked from each of J101-E5, J101-31 and J101-E7 plates, inoculated in LB + Cm 12.5 and grown.