Protease and Proteinase Trials
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.
Image 1: 23chr, 10min incubation, 1uL protease added
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