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From 2010.igem.org

Contents 1 July 12th 2 July13th 3 July 14th 4 July 15th 5 July 16th

July 12th

• Grow more:

  -B1002(terminator)
  -K112808
  -CI promoter
  -LuxR

• Digests:

  -J24679+B1002(D+B) (XP+phosphatase)
  -J24679+B1006(A+C) (XP+phosphatase)
  -C0261 (SP)
  -K274100 (ES)
  -B1002 (EX)
  -B1006 (EX)

• Ligations:

  Insert       Vector

1)J24679+B1002 C0261 2)J24679+B1006 C0261 3)K274100 B1002

                B1006

• Transform:

   -R0051 from kit plate 2009

• Gel order:

  -Ladder
  -B1006
  -B1002
  -K274100 (July 1st)
  -K274100 (June 22nd)
  -C0261

• All the bands showed up in the correct spots and so they were all cut out and gel purified.

July13th

• Colony PCR

 - J24679+B1006+C0261(C) (A-D)
 - J24679+B1006+C0261(A) (A-D)
 - K274100+B1002 (A-D)
 - K274100+B1006 (A-C)
 - J24679+B1002+C0261(D) (A-D)
 - J24679+B1002+C0261 (B)(A-D)

• Digests:

 -R0010+P0451 (ES+phosphatase)
 -R0010 (EX)

• Ligations:

 -R0010+P0451

• Gel Order:

 -Ladder
 -R0051
 -1A-D
 -2A-D
 -5A-D

• Gel Order:

 -Ladder
 -Lux
 -3A-D

 -Ladder
 -4A-C
 -6A-D

The ones that are marked in the picture above tells us that 6A and 6C were grown up.

July 14th

• Digests:

 -K274100 (ES)
 -B1002 (XP+Phosphatase)
 -B1006 (XP+Phosphatase)
 -C0062 (EP)
 -K3 (EP)
 -R0010 (ES+Phosphatase)
 -P0451 (XP)
 -J24679 (ES)
 

• Ligations:

 -K274100+B1002
 -K274100+B1006
 -C0062+K3
 -C0062+B1002
 -C0062+B1006
 -R0010+P0451
 -J24679+B1002
 -J24679+B1006

• Gel Order:

 -Ladder
 -J24679
 -K274100
 -P0451
 -A3
 -K3

All the bands appeared where we wanted them to be and so all 5 bands were cut out and then gel purified.

July 15th

• Colony PCR:

  -K274100+B1002 (A-C)
  -K274100+B1006 (A-C)
  -C0062+B1002 (A-C)
  -J24679+B1002 (A-C)
  -J24679+B1006 (A-C)
  -C0062+B1006 (A-C)

• Digests:

  -C0062+RBS (EP)
  -R0010 (ES+Phosphatase)
  -J23100 (ES+Phosphatase)
  -B1006 (XP+Phosphatase)
  -B1002 (XP+Phosphatase)
  -K3 (EP)
  -A3 (EP)
  -P0451 (XP)
  -PJ241 (XP)
  -PJ241 (ES)

• Gel Order:

  - Ladder
  - PJ241(ES)
  - PJ241(XP)
  - P0451 
  - C0261+RBS
  -A3
  -K3

Although the band for C0261+RBS was faint, we were still able to cut it out. All the other bands showed up nicely and were also cut and then gel purified.

Colony PCR was ran on this gel:

The colonies 3B, 4A, 4C,and 5C showed up very well and so they were sent out to get sequenced.

July 16th

• Digests:

 -RBS+C0062 (EP)
 -A3 (EP)
 -C0062 (ES)
 -J24679 (ES)
 -K3 (EP)
 -B1006 (XP+Phosphatase)
 -B1002 (XP+Phosphatase)

• Ligations:

 -RBS+C0062+A3
 -C0062+B1006
 -C0062+B1002
 -J24679+B1006
 -J24679+B1002
 -J24679+K3
 -C0261+K3

• Gel Order:

 -Ladder
 -A3
 -K3
 -J24679
 -Lux+RBS

After the gel was ran for about 40-45 minutes, all the bands appeared around where they should have shown up and so they were all cut out and then gel purified to be used in the ligations.

Colony PCR was ran on this gel.

 -P0451+R0010

• Gel Order:

 -Ladder
 -P0451+R0010 (A)
 -P0451+R0010 (B)
 -P0451+R0010 (C)
 -P0451+R0010 (D)

Since only A and D showed up, they were grown up.

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