Protease and Proteinase Trials

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Revision as of 21:28, 12 April 2010 by >Jbrubake (→‎Setup)
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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

Results

To conserve resources, only the 23 chromosome wells were imaged. Any effects on 23 chromosome wells were assumed to have had similar effects on the 12 chromosome wells.

File:23chr 10min 1uL.jpg

Image 1: 23chr, 10min incubation, 1uL protease added

File:23chr 20min 1uL.jpg

Image 2: 23chr, 20min incubation, 1uL protease added

Discussion

Protease had no noticeable effect on the chromosomes. Two likely reasons for this: 1) short incubation time. 2) not enough protease added. Also, had no frame of reference because a positive control was not added into this trial (i.e. 0.5mg/mL Proteinase K)

4/3/10

Setup

One strip was used.

         | 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

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