Purpose: The purpose of this lab was to finish the last few steps from the previous protocol and examining our pellet. The next purpose was to try a new protocol named EZNA tissue DNA Kit Protocol. The purpose of using this protocol is because it seemed to have very successful results, so we are trying to replicate that success. This protocol involved the use of many new substances such as ethanol and buffers.

Procedure:

• Remove supernatant from test tube containing cell pellet
• Watch cells with 200 microliters of PBS
• Remove the PBS and put another fresh 200 microliters of PBS
• Place 5 drops of microliters on a concavity slide and then place 20microliter of iodine in the drops
• Examine the drops under a microscope and conduct cell counts
• Add 25 microliter of protease solution and vortex
• Add 220 microliter of BL Buffer which aids in denaturing proteins
• Incubate tube at 70 degrees Celsius for 5 minutes in a heating block, and then vortex briefly
• Add 22o microliter of 100% ethanol, and then vortex thoroughly
• label tubes and store in freezer

Data and Observations

• Unfortunately, my group was only able to complete up to step 8 of the procedure due to complications at the beginning of the procedure
  • The pellet from last week never accumulated, so we had to remove the supernatant from the tube and then add PBS
  • After adding the PBS, we had to centrifuge test tube again to produce a pellet
  • Pellet still did not completely isolate
  • Steps to remove the supernatant were repeated
  • Began the assigned protocol 20 minutes after other groups had started
  • Fortunately, the pellet we collected was fairly large and easy to analyze
• Cell Counts for 2 microliter drops
  • Drop 1: 20
  • Drop 2: 7
  • Drop 4: 16
  • Drop 5: 24
• The test tube is labeled 21-1 TCA and the protocol will be continued through open lab

Conclusion

Unfortunately, my group was not able to finish the protocol and see if the results were for accurate than previous protocols. However, from the literature review about this protocol, I have a good amount of hope that it will be a better protocol than past ones. In the future, I know my group hopes to be able to complete these protocols within the timeline of the scheduled lab time. We need to be very punctual, especially because vortexing and centrifuging take a lot of time.