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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== 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== ===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: 23chr_10min_1uL.jpg]] ''Image 1: 23chr, 10min incubation, 1uL protease added'' [[image: 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 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=== 27C, 22h30min ===Results=== 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: [[image: 23chr_2uL_PK.jpg]] ''Image 1: 23chr, 2uL Proteinase K'' [[image: 23chr_2uL_P.jpg]] ''Image 2: 23chr, 2uL Protease'' ===Discussion=== 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== ===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 ===Results=== '''Non-denatured samples:''' [[image: 12chr_PK.jpg]] ''Image 1: 12 chromosome, 0.5uL Proteinase K'' [[image: 23chr_8uL_P.jpg]] ''Image 2: 23 chromosome, 8uL Protease'' '''Denatured samples:''' [[image: preheat_12chr_PK.jpg]] ''Image 3: Denatured - 12 chromosome, 0.5uL Proteinase K'' [[image: preheat_23chr_6uL_P.jpg]] ''Image 4: Denatured - 23 chromosome, 6uL Protease'' [[image: preheat_23chr_8uL_P.jpg]] ''Image 5: Denatured - 23 chromosome, 8uL Protease'' ===Discussion=== 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== ===Setup=== 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=== 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=== 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. [[image: 4-22-10_PK_CTRL.jpg]] ''Image 1: 0.5uL Proteinase K'' [[image: 4-22-10_6uL_Protease.jpg]] ''Image 2: 6uL Protease'' [[image: 4-22-10_8uL_Protease.jpg]] ''Image 3: 8uL Protease'' [[image: 4-22-10_10uL_Protease(1).jpg]] ''Image 4: 10uL Protease, immediately after exposure'' [[image: 4-22-10_10uL_Protease(2).jpg]] ''Image 5: 10uL Protease, waiting ~15 seconds after exposure'' [[image: 4-22-10_2uL_Prep.jpg]] ''Image 6: 2uL PrepGEM'' [[image: 4-22-10_3uL_Prep.jpg]] ''Image 7: 3uL PrepGEM'' [[image: 4-22-10_4uL_Prep.jpg]] ''Image 8: 4uL PrepGEM'' ===Discussion=== 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== ===Setup=== 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=== 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=== *All samples were stained with 3uL 2X Dapi dye, regardless of total volume From '''Strip 1''' [[image: Neg_10X_4-30-10.jpg]] ''Image 1: Negative control, 10X'' [[image: Tube5_1uL_PK_10X_4-30-10.jpg]] ''Image 2: Positive control - 1uL P.K., 10X'' [[image: Tube5_10uL_P_10X_4-30-10.jpg]] ''Image 3: 10uL P., 10X'' [[image: Tube5_12uL_P_10X_4-30-10.jpg]] ''Image 4: 10uL P., 10X'' [[image: Tube5_15uL_P_10X_4-30-10.jpg]] ''Image 5: 15uL P., 10X'' From '''Strip 2''' [[image: Neg_10X_5-1-10.jpg]] ''Image 1: Negative control, 10X'' [[image: Tube5_2uL_Prep_10X_5-1-10.jpg]] ''Image 7: 2uL Prep., 10X'' [[image: Tube5_3uL_Prep_10X_5-1-10.jpg]] ''Image 8: 3uL Prep., 10X'' [[image: Tube5_4uL_Prep_10X_5-1-10.jpg]] ''Image 9: 4uL Prep., 10X''
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