Protease and Proteinase Trials: Difference between revisions

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The volumes of protease or proteinase K were the same in both strips:
The volumes of protease or proteinase K were the same in both strips:


Rows 1 and 2: Nothing
Rows 1 and 2: Nothing
Row 3: 0.5uL P.K. (Positive Control)
Row 3: 0.5uL P.K. (Positive Control)
Row 4: 2uL P
Row 4: 2uL P
Row 5: 4uL P
Row 5: 4uL P
Row 6: 6uL P
Row 6: 6uL P
Row 7: 8uL P
Row 7: 8uL P
Row 8: Nothing (Negative Control)
Row 8: Nothing (Negative Control)


===Incubation===
===Incubation===

Revision as of 20:55, 12 April 2010

Two different denaturants were used:

  • Protease (Qiagen) - abbreviated P.
  • Proteinase K (Invitrogen) - abbreviated P.K.

Solution Preparations

The protease (Qiagen) was easily reconstituted: 7uL water per vial

See Proteinase K Protocol for preparation of Proteinase K (Invitrogen)

3/31/10

Setup

2 columns/strips of sorted chromosomes were collected from Sam. The strips were separated as follows:

          | H | - Head
Row 1     | o | - No chromosomes
Row 2     | o | - 1 chromosome
Row 3     | o | - 12 chromosomes
Row 4     | o | - 12 chromosomes
Row 5     | o | - 12 chromosomes
Row 6     | o | - 23 chromosomes
Row 7     | o | - 23 chromosomes
Row 8     | o | - 23 chromosomes
          | A | - Tail

Protease was added as follows:

Rows 1,2,5,8: No P. added
Rows 3,6: 1uL P. added
Rows 4,7: 0.5uL P. added

The same was applied to both strips, labelled Strip 1 and Strip 2

Incubation

Strip 1: 37C, 10min -> 70C, 15min Strip 2: 37C, 20min -> 70C, 15min

4/9/10

Setup

2 columns/strips of sorted chromosomes were collected from Sam. The strips were separated as follows:

          | H | - Head
Row 1     | o | - No chromosomes
Row 2     | o | - 1 chromosome
Row 3     | o | - 12 chromosomes
Row 4     | o | - 12 chromosomes
Row 5     | o | - 12 chromosomes
Row 6     | o | - 23 chromosomes
Row 7     | o | - 23 chromosomes
Row 8     | o | - 23 chromosomes
          | A | - Tail

The volumes of protease or proteinase K were the same in both strips:

Rows 1 and 2: Nothing
Row 3: 0.5uL P.K. (Positive Control)
Row 4: 2uL P
Row 5: 4uL P
Row 6: 6uL P
Row 7: 8uL P
Row 8: Nothing (Negative Control)

Incubation

Strip 1: Heat denatured before adding proteases

95C, 10min -> 25C, 2min
*Remove and add appropriate reagents (P. and P.K.)
37C, 23h36min

Strip 2: No heat denaturing

37C, 23h36min