Week 4: New Plasmid

 Introduction 

    Since our failed plasmid extractions last week with our kit, GeneJET Mini Prep, we decided to switch to the ZymoPure Plasmid Kit instead. We hoped that the new kit procedure would help us achieve our desire concentration for this extraction. Our wanted concentration is 30 ul. Below is our procedure for the new ZymoPure Plasmid Kit that we spent this week doing. 

Process

    Before we start on the kit, we have to centrifuge a total of 3mL of culture in tubes A, B, Ae, and Be and incubate it in 500uL of multibuffer 2 times for 5 minutes. Then, we would incubate it in 600 uL of lysozyme for 45 minutes at room temperature. (Initially we did 30 minutes but it was not enough) After doing this process, we are able to move onto the kit. The kit has a different kind of protocol than the previous kit. For this kit, instead of starting with resuspension and supernatant, we used the ZymoPure P1, P2, and P3 individually and resuspending completely every time we would use each solution. Not only that, towards the end of our protocol, we used the ZymoPure wash 1 and wash 2 (wash 2-2x). After the whole protocol, we store the samples below and around -20 C to keep the DNA contacted. 

Results

    Unfortunately, the results seems to be extremely off. We nanodropped the samples below. 

ng/ul A260/280 A260/230

A 3.3    15.74         -1.65

B 6.2          2.04          4.33

Ae    17.6          1.94            2.00

Be 87.7          1.79                1.77

Discussion

    Obviously these results are not good enough. We must retry the whole protocol again. After discussing the possible errors for this extraction, we might believe that it could be over lyzoming the samples way too much (lyzome incubation + red solution), common user error, or chromosomal crossover.

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