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)
 
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[[Image:Labnotes html m7c8f4d24.jpg|300px|thumb|left|K2 K1]]
[[Image:Labnotes html m7c8f4d24.jpg|300px|thumb|left|K2 K1]]
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[[Image:Labnotes html 7746e05a.jpg|300px|thumb|K3]]
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<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>
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</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.

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