Choi, James_Report_Digestion Analysis

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Biology

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Apr 3, 2024

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James Choi MCB 251 Digestion Analysis Procedure : 1. Obtain a gel electrophoresis chamber, a casting tray, and an agarose gel (with the wells facing the negative end). Fill the chamber with a buffer so that it is above the gel and covering the wells. 2. Obtain the DNA restriction enzyme made last week. 3. Add 10 uL of 5x blue to each microfuge tube, and pipette gently to thoroughly mix. 4. Using a P20 micropipette, insert them into the gel using the following measurements. a. Lane #1 (10 uL): DNA ladder b. Lane #2 (15 uL): EcoRV (E) c. Lane #3 (15 uL): PstI (P) d. Lane #4 (15 uL): EcoRI + PstI (EP) e. Lane #5 (15 uL): Undigested (C) f. Lane #6 (15uL): BamHI (B) g. Lane #7 (2 uL): DNA ladder 5. Set the P20 to 10uL and carefully add the ladder, then after changing tips set the P20 to 15uL and add each DNA sample. Be careful not to puncture the gel. 6. Remain other wells empty. 7. Place the lid onto the gel electrophoresis chamber, and plug in the electrodes to the power box. Before applying the current, wait for all the lab groups. 8. Run the gel at 100V for 90 minutes, or until the bands are halfway across the gel. Check for bubbles in the buffer. 9. Once the process is complete, cut the power, remove the electrodes, and obtain a casting tray with gel.

10. Bring it to the UV transilluminator. 11. Gently slide it off the tray into the box and make sure the lamps are off. 12. Close the machine and capture an image of the gel, which should be fluoresced due to the UV lamps lighting the ethidium bromide. Data/Results: The result of our gel electrophoresis was unclear due to the lack of band size. This might be caused by the low quantity of the sample. Therefore, we are going to use demo gel for pBR322 digestion. Demo gel for the pBR322

For Demo gel, each lane contains: - Lane #1: Ladder - Lane #2: BamHI - Lane #3: EcoRI - Lane #6: EcoRI + PstI - Lane #7: PstI Lane 1: Ladder (Standard) Lane 2: BamHI Distant traveled (mm) Base Pairs (kb) 28.5 4.3 Lane 3: EcoRI Distant traveled (mm) Base Pairs (kb) 28 4.5 Distant traveled (mm) Base Pairs (kb) 20 10.0 22 8.0 24.5 6.0 26.5 5.0 29 4.0 34 3.0 40.5 2.0 46 1.5 55 1.0 68 0.5

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Lane 6: EcoRI +PstI Distant traveled (mm) Base Pairs (kb) 32 3.2 Lane 7: PstI Distant traveled (mm) Base Pairs (kb) 28 4.5 For Bp calculation of BamHI digest: x = 28.5, 26422*e^(-0.06*28.5) = 4778.84 bps Discussion: Because our gel had unclear image of DNA band due to lack of quantity of the sample, we used demo gel pBR322 to analyze the data. Our gel did not show a clear image probably because of a loading issue of our sample or lack of quantity of the sample. Still, some of the bands showed so we conclude that there were no errors made in the Qiagen procedure used to extract the plasmid.

1. Demo gel results indicated that restriction enzymes functioned properly in lanes 2, 3, 6, and 7. Comparing the plasmid map of PBR322, we got valid results on gel due to similar base pair size by using restriction enzymes. For instance, single digestion, which is lanes 2, 3, and 7, showed a similar size of the base pairs since they only have a 1 cut site that makes a single linear plasmid. For double digestion, which is lane 6, showed about 3200 base pairs; however, for exact calculations, EcoRI cuts at 4359 and PstI cuts at 3611. 4359 - 3611 = 748, and 4361 (entire size of pBR322) - 748 = 3613 bps, which shows a similar size compared to what demo gel electrophoresis showed. 2. The graph is shown in the data/results section. Based on pBR322 plasmid map, the base pair length was supposed to be 4361 and should be the same after the cut changes it from circular to linear. However, our observation of the size of the base pair had some differences from the graph's best trend line. For example, the total size of pBR322 should be 4361 bps similar to when it was cut by EcoRI, but when we put data of EcoRI lane to the function, it showed 26422*e^(-0.06*28) = 4924 bps, which exceeds the total base pair of pBR322. It is possible that the restriction enzymes did not cut the demo’s plasmids and therefore left them as circles, and the plasmids were supercoiled which would allow them to travel down the gel faster resulting in more base pair sizes because it is being compared to linear DNA.

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