Team:Uppsala-SwedenWeek15

From 2010.igem.org

(Difference between revisions)
(Plasmid concentration of K1, K2, K3, (old version) and digestion)
(Plasmid concentration of K1, K2, K3, (old version) and digestion)
 
(5 intermediate revisions not shown)
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</TR>
</TR>
</TABLE>
</TABLE>
 +
 +
 +
E= EcorI
 +
P= PstI
 +
FDB= Fast Digest Buffer
 +
 +
 +
Gel of digested samples with 0.8% agarose gel . picture missing
 +
 +
Counting from the ladder. K1(Colony 1-5) and K2(colony 1-6)
 +
 +
[[Image:Labnotes html m7c8f4d24.jpg|300px|thumb|left|K2 K1]]
 +
 +
[[Image:Labnotes html 7746e05a.jpg|300px|thumb|K3]]
 +
 +
 +
<TABLE WIDTH=596 BORDER=1 BORDERCOLOR="#000000" CELLPADDING=7 CELLSPACING=0>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">Sample</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" STYLE="margin-bottom: 0in">DNA</P>
 +
<P CLASS="western"><SPAN LANG="sv-SE">(µL/ml)</SPAN></P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P CLASS="western"><SPAN LANG="sv-SE">DNA 100X)</SPAN></P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western">DNA required</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western">Enzymes (E &amp; P)</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">FDB (10X)</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">H20</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">Total (µL)</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K1C1</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western">0.941</P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">94.1</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>5.3</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">1</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">2.7</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">20</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K1C2</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western">0.670</P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">67.0</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>7.4</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">1</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">0.6</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">20</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K1C3</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western">0.736</P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">73.6</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>6.7</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">1</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P CLASS="western"><SPAN LANG="sv-SE">1.3</SPAN></P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">20</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K1C4</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western">0.522</P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">52.2</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>9.5</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">2</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">7.5</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">10</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K1C5</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P CLASS="western"><SPAN LANG="sv-SE">0.622</SPAN></P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">62.2</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>8.0</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">2</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">9.0</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">10</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K2C1</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P CLASS="western"><SPAN LANG="sv-SE">0.806</SPAN></P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">80.6</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>6.2</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">1</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">1.8</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">10</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K2C2</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western">0.567</P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">56.7</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>8.8</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">2</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">2.6</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">10</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K2C3</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western">0.611</P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">61.1</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>8.1</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">2</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">3.7</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">10</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K2C4</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western">0.671</P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">67.1</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>7.4</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">1</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">6.1</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">20</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K2C5</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P CLASS="western"><SPAN LANG="sv-SE">0.828</SPAN></P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">82.8</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>6.0</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">1</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">6.6</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">10</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K3C1</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western">0.678</P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">67.8</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>7.3</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">1</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">2.6</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">10</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K3C2</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western">0.955</P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">95.5</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>5.2</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">1</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">2.4</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">10</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K3C3</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P CLASS="western"><SPAN LANG="sv-SE">0.841</SPAN></P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">84.1</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>5.9</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">1</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">3.5</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">10</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K3C4</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western">1.11</P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">111.0</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>4.5</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">1</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">3.8</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">10</P>
 +
</TD>
 +
</TR>
 +
<TR VALIGN=TOP>
 +
<TD WIDTH=49>
 +
<P LANG="sv-SE" CLASS="western">K3C5</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P CLASS="western"><SPAN LANG="sv-SE">0.868</SPAN></P>
 +
</TD>
 +
<TD WIDTH=64>
 +
<P LANG="sv-SE" CLASS="western">86.8</P>
 +
</TD>
 +
<TD WIDTH=52>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>5.7</P>
 +
</TD>
 +
<TD WIDTH=109>
 +
<P LANG="sv-SE" CLASS="western" ALIGN=CENTER>0,5 &amp;0.5</P>
 +
</TD>
 +
<TD WIDTH=62>
 +
<P LANG="sv-SE" CLASS="western">1</P>
 +
</TD>
 +
<TD WIDTH=33>
 +
<P LANG="sv-SE" CLASS="western">3.5</P>
 +
</TD>
 +
<TD WIDTH=61>
 +
<P LANG="sv-SE" CLASS="western">10</P>
 +
</TD>
 +
</TR>
 +
</TABLE>
 +
 +
[[Image:Labnotes html m15593582.jpg|400px|thumb|left|K1 K2]] [[Image:Labnotes html m4d07be2b.jpg|400px|thumb|right|K2 K3]]
 +
 +
 +
The K4, K5 and K6 construct are composed of J1, J2 and J3 assembled with BB_I0460 which contains both Ampicillin and Kanamycin cassettes in its backbone, psB1AK3, and the J- construct contains Chloramphinicol cassette, psB1C3. The Tetracyclin antibiotic hasn’t work as expected. Therefore the 3A assembly technique wouldn’t be possible to apply due there is no antibiotics left available with a corresponding backbone.
 +
 +
One option to get around this obstacle was to run the gel, after digestion of BB_I04060, and cut the band with the Biobrick-length and then 3A assembly it with the J-construct and E-P digested psB1C3.
 +
 +
Another option was to switch backbone of J-construct to psB1C3 and then use 3A-assembly it with BB_I0460 and E-P digested psB1C3. We used the second method because it’s a more safer way even though it takes longer time to finish.
 +
 +
 +
Ligation of: J1 = I1+H6 , J2 = I2 + H3, J3 = I3 + H8.to a psB1C3 – vector.
 +
Transformation and incubation overnight.

Latest revision as of 19:50, 26 October 2010

Week-15

Plasmid concentration of K1, K2, K3, (old version) and digestion

Sample

DNA-concentration

(µL/ml)

DNA

Enzymes (E & P)

FDB (10X)

H20

Total (µL)

K1C1

32,7

15.3

0,5 &0.5

2

1.7

20

K1C2

98.3

5.0

0,5 &0.5

2

12

20

K1C3

74.4

6.7

0,5 &0.5

2

10.3

20

K1C4

516.4

0.9

0,5 &0.5

1

7.1

10

K1C5

135.4

3.6

0,5 &0.5

1

4.4

10

K2C1

161.7

3.1

0,5 &0.5

1

4.9

10

K2C2

92.3

5.4

0,5 &0.5

1

2.6

10

K2C3

112.2

4.3

0,5 &0.5

1

3.7

10

K2C4

45.6

10.9

0,5 &0.5

2

6.1

20

K2C5

348.1

1.4

0,5 &0.5

1

6.6

10

K2C6

91.3

5.4

0,5 &0.5

1

2.6

10

K3C1

88.4

5.6

0,5 &0.5

1

2.4

10

K3C2

109.9

4.5

0,5 &0.5

1

3.5

10

K3C3

116.6

4.3

0,5 &0.5

1

3.8

10

K3C4

110.0

4.6

0,5 &0.5

1

3.5

10

K3C5

90.67

5.5

0,5 &0.5

1

2.5

10

K3C6

109.5

4.6

0,5 &0.5

1

3.4

10


E= EcorI P= PstI FDB= Fast Digest Buffer


Gel of digested samples with 0.8% agarose gel . picture missing

Counting from the ladder. K1(Colony 1-5) and K2(colony 1-6)

K2 K1
K3


Sample

DNA

(µL/ml)

DNA 100X)

DNA required

Enzymes (E & P)

FDB (10X)

H20

Total (µL)

K1C1

0.941

94.1

5.3

0,5 &0.5

1

2.7

20

K1C2

0.670

67.0

7.4

0,5 &0.5

1

0.6

20

K1C3

0.736

73.6

6.7

0,5 &0.5

1

1.3

20

K1C4

0.522

52.2

9.5

0,5 &0.5

2

7.5

10

K1C5

0.622

62.2

8.0

0,5 &0.5

2

9.0

10

K2C1

0.806

80.6

6.2

0,5 &0.5

1

1.8

10

K2C2

0.567

56.7

8.8

0,5 &0.5

2

2.6

10

K2C3

0.611

61.1

8.1

0,5 &0.5

2

3.7

10

K2C4

0.671

67.1

7.4

0,5 &0.5

1

6.1

20

K2C5

0.828

82.8

6.0

0,5 &0.5

1

6.6

10

K3C1

0.678

67.8

7.3

0,5 &0.5

1

2.6

10

K3C2

0.955

95.5

5.2

0,5 &0.5

1

2.4

10

K3C3

0.841

84.1

5.9

0,5 &0.5

1

3.5

10

K3C4

1.11

111.0

4.5

0,5 &0.5

1

3.8

10

K3C5

0.868

86.8

5.7

0,5 &0.5

1

3.5

10

K1 K2
K2 K3


The K4, K5 and K6 construct are composed of J1, J2 and J3 assembled with BB_I0460 which contains both Ampicillin and Kanamycin cassettes in its backbone, psB1AK3, and the J- construct contains Chloramphinicol cassette, psB1C3. The Tetracyclin antibiotic hasn’t work as expected. Therefore the 3A assembly technique wouldn’t be possible to apply due there is no antibiotics left available with a corresponding backbone.

One option to get around this obstacle was to run the gel, after digestion of BB_I04060, and cut the band with the Biobrick-length and then 3A assembly it with the J-construct and E-P digested psB1C3.

Another option was to switch backbone of J-construct to psB1C3 and then use 3A-assembly it with BB_I0460 and E-P digested psB1C3. We used the second method because it’s a more safer way even though it takes longer time to finish.


Ligation of: J1 = I1+H6 , J2 = I2 + H3, J3 = I3 + H8.to a psB1C3 – vector. Transformation and incubation overnight.