Week of 6/28

From 2010.igem.org

(Difference between revisions)

Revision as of 14:35, 5 August 2010

June 28

Retransform:

  -R0061
  -R0062
  -R0063
  -K091146

Inventory

-Good Sequencing:
    o Backbone - C3RFP
    o pLux     - K091146
    o Lysis    - K112022
    o pLux     - R0063
    o pLux     - R1062
    o CI+RBS   - K081007
    o Cons.Prom- J23116
    o Cons.Prom- J23108
    o Pigment  - K274100
    o LuxI     - C0261
    o Lysis    - K112808
    o pLac     - R0010
    o LacI+RBS - J24679

To grow:

Electrocompetent cells were made.

June 29

Gel Order:

   - J23108
   - J23108 (Digest 2)
   - R0063  
   - R0063  (Digest 2)
   - K081007
   - K081007(Digest 2)
   - J23116 
   - J23116 (Digest 2)

Gel extract was done using Digest number 1. In this gel we ran the same part twice but from two different digests. Digest 1 was a 6 hour digest in the 37 degree Celsius water bath while Digest 2 was a 1 hour digest in the same water bath. As you can see, the results do not differ all that noticeably. That being established we went back to doing 1 hour digests.

The bands that were cut out are provided below:

All these bands were purified.

June 30

On this gel, we ran the Colony PCR and the digests. The order is as follows:

Colony PCR:

  - Ladder
  - A-J

Digests:

  - Ladder
  - K091107 (digest 2)
  - K091107 
  - K091146 (digest 2)
  - K091146
  - J23116+J37033 (digest 2)
  - J23116+J37033
  - R0010 (digest 2)
  - R0010

The bands that were cut are marked with a square around them in the picture above. They were also then purified.

3 bands that were cut:

   - K091107
   - K091146
   - R0010

A second gel was ran the same day. The order is provided below:

  - Ladder
  - B1002
  - Lux R 2
  - C0261+J24679

As you can see, the only 2 bands that were cut were B1002 and C0261+J24679

Grow for tomorrow:

  - K274100
  - K112022

July 1

Flasks minipreped:

   - B1006
   - K274100
   - J23100

Digests:

   - B1006 (EX)
   - K274100 (XP)
   - J23100 (SP)

Sent to the sequencing center:

  1) B1002
  2) B1006
  3) S01414
  4) J23100
  5) C0261+J24679

Gel order:

  - Ladder
  - B1006
  - K274100
  - J23100

All the bands appeared where we wanted them to so we cut out all three bands and then purified them.

Ligations:

  Vector        Inserts

1)J23100 K274100 2)J23100 S01414 3)R0063 a)K274100 4)K091146 b)C0261+J24679 5)K091107 6)R0062 7)R0010 K081007

Tomorrow:

- Develop primers to figure out if K274100 and K112808 are correct.

July 2

Colony PCR's ran on gel.The order is as follows:

   - Ladder
   - 1A, 1B, 1C
   - 2A, 2B, 2C
   - 3A, 3B, 3C

   - Ladder
   - 4A, 4B, 4C
   - 5A, 5B, 5C
   - 6A, 6B, 6C
   - 7A, 7B, 7C

Unfortunately, we accidentally put ladder in all the Colony products. The picture is provided above.

@@ Line 47: / Line 47: @@
 <center>[[Image:6-29-10.jpg]]</center>
-*Gel extract was done using Digest number 1.
+*Gel extract was done using Digest number 1. In this gel we ran the same part twice but from two different digests. Digest 1 was a 6 hour digest in the 37 degree Celsius water bath while Digest 2 was a 1 hour digest in the same water bath. As you can see, the results do not differ all that noticeably. That being established we went back to doing 1 hour digests.
 *The bands that were cut out are provided below:
@@ Line 56: / Line 56: @@
 All these bands were purified.
 ==June 30==