Protease and Proteinase Trials

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Three different denaturants were used:

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


Solution Preparations[edit]

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

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

See PrepGEM (ZyGEM) Preparation for preparation of PrepGEM (ZyGEM)

3/31/10[edit]

Setup[edit]

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[edit]

Strip 1:

37C, 10min -> 70C, 15min

Strip 2:

37C, 20min -> 70C, 15min

Results[edit]

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[edit]

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[edit]

Setup[edit]

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

Incubation[edit]

27C, 22h30min

Results[edit]

The slide was prepared incorrectly (used too much volume): some of the samples mixed, as evident by denatured strands in the negative control. Still, clear images of each sample were found:

File:23chr 2uL PK.jpg

Image 1: 23chr, 2uL Proteinase K

File:23chr 2uL P.jpg

Image 2: 23chr, 2uL Protease

Discussion[edit]

It is clear that the Proteinase K is more effective than the Protease solution. The incubation time is sufficient, as noted by each manufacturer. In order to try and close the gap between Proteinase K and Protease, different amounts of Protease need to be assessed.

4/9/10[edit]

Setup[edit]

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[edit]

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

Results[edit]

Non-denatured samples:

File:12chr PK.jpg

Image 1: 12 chromosome, 0.5uL Proteinase K

File:23chr 8uL P.jpg

Image 2: 23 chromosome, 8uL Protease

Denatured samples:

File:Preheat 12chr PK.jpg

Image 3: Denatured - 12 chromosome, 0.5uL Proteinase K

File:Preheat 23chr 6uL P.jpg

Image 4: Denatured - 23 chromosome, 6uL Protease

File:Preheat 23chr 8uL P.jpg

Image 5: Denatured - 23 chromosome, 8uL Protease

Discussion[edit]

While the results of this trial were somewhat less clear than previous trials, it appears that preheating and denaturing the samples before adding Protease aids in the denaturing capabilities of the Protease solution. On top of this, the difference between 6uL and 8uL of Protease is pretty small. To get higher amounts of Protease without overwhelming the volume of the chromosomes would be to reconstitute another vial with less water, increasing the concentration. This way we could test the effects of much higher amounts of protease without increasing the volume too much.

Recommendation for future trials: Continue to preheat the samples before adding Protease, and use a more concentrated Protease solution.

4/20/10[edit]

Setup[edit]

A full strip of PCR tubes was used, but rather than using the sorted chromosomes, the filtered tubes 1+2 sample was used. 3uL sample per well. To each well the following proteases were added:

Row 1 - 0.5uL P.K.
Row 2 - 6uL P.
Row 3 - 8uL P.
Row 4 - 10uL P.
Row 5 - 2uL Prep.
Row 6 - 3uL Prep.
Row 7 - 4uL Prep.
Row 8 - Negative Control

Incubation[edit]

37C, 23h

  • Problems: 1) Forgot to pre-heat the samples. 2) Failed to follow the correct heating instruction for the PrepGEM protease -> PrepGEM failed

Results[edit]

All of the wells were stained with 3uL 2X SYBR Green, regardless of volume of sample in the well. To be consistent, a maximum of 5uL was used from each well and plated.

File:4-22-10 PK CTRL.jpg

Image 1: 0.5uL Proteinase K

File:4-22-10 6uL Protease.jpg

Image 2: 6uL Protease

File:4-22-10 8uL Protease.jpg

Image 3: 8uL Protease

File:4-22-10 10uL Protease(1).jpg

Image 4: 10uL Protease, immediately after exposure

File:4-22-10 10uL Protease(2).jpg

Image 5: 10uL Protease, waiting ~15 seconds after exposure

File:4-22-10 2uL Prep.jpg

Image 6: 2uL PrepGEM

File:4-22-10 3uL Prep.jpg

Image 7: 3uL PrepGEM

File:4-22-10 4uL Prep.jpg

Image 8: 4uL PrepGEM

Discussion[edit]

As previously mentioned, the PrepGEM failed because the heating cycle recommended for that protease was not followed. The images show only stained, non-denatured samples.

The Protease appears to be working at higher volumes, but as is evident between the two different 10uL Protease images, it is hard to tell exactly what the result of the denaturing incubation was. This is because SYBR Green degrades rapidly once introduced to light. The next trial will use Dapi as the staining dye.

4/30/10[edit]

Setup[edit]

Two strips were used. The strip with the PrepGEM protease had to be treated differently than the other strip, as noted in the Incubation section. Each well contained 3uL of sample from Tube 5 and to each well the following proteases were added:

Strip 1:

Row 1 - Negative Control
Row 2 - 1uL P.K.
Row 3 - 10uL P.
Row 4 - 12uL P.
Row 5 - 15uL P.

Strip 2:

Row 1 - Negative Control
Row 2 - 2uL Prep.
Row 3 - 3uL Prep.
Row 4 - 4uL Prep.

Incubation[edit]

Both strips were pre-heated at 90C for 10min to help denature the samples. After the pre-heating:

Strip 1

37C, 22h

Strip 2

75C, 10min -> 15C, 15min

Results[edit]

  • All samples were stained with 3uL 2X Dapi dye, regardless of total volume

From Strip 1

File:Neg 10X 4-30-10.jpg

Image 1: Negative control, 10X

File:Tube5 1uL PK 10X 4-30-10.jpg

Image 2: Positive control - 1uL P.K., 10X

File:Tube5 10uL P 10X 4-30-10.jpg

Image 3: 10uL P., 10X

File:Tube5 12uL P 10X 4-30-10.jpg

Image 4: 10uL P., 10X

File:Tube5 15uL P 10X 4-30-10.jpg

Image 5: 15uL P., 10X


From Strip 2

File:Neg 10X 5-1-10.jpg

Image 1: Negative control, 10X

File:Tube5 2uL Prep 10X 5-1-10.jpg

Image 7: 2uL Prep., 10X

File:Tube5 3uL Prep 10X 5-1-10.jpg

Image 8: 3uL Prep., 10X

File:Tube5 4uL Prep 10X 5-1-10.jpg

Image 9: 4uL Prep., 10X