Protease and Proteinase Trials: Difference between revisions
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>Jbrubake (→Setup) |
>Jbrubake |
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===Incubation=== | ===Incubation=== | ||
Strip 1: 37C, 10min -> 70C, 15min | Strip 1: | ||
Strip 2: 37C, 20min -> 70C, 15min | 37C, 10min -> 70C, 15min | ||
Strip 2: | |||
37C, 20min -> 70C, 15min | |||
==4/9/10== | ==4/9/10== |
Revision as of 20:59, 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