Week 8 2/26-3/1: E.coli plasmid extraction

 Introduction 

    We begin our experiment this week by preparing the correct media to grow both of our E.coli and D.caeni. Luckily, E.coli only takes a day to grow on both plates and broth so we were able to get started on our plasmid extraction earlier in the week. While we are running our extraction, we are also growing our D.caeni in plates and broth since it was known that the cells grew slower. Below are the steps we took to run our plasmid extraction to get pRad 1 from E.coli cells. 

Procedure

Before we perform plasmid extraction, we had to OD value our E.coli broth sample and gram-stain the cells to find out if the samples are fresh and clean. We are also going to run 4 reactions. To do this, we would need 8 ephindorph tubes (4 for set #1 and 4 for set #2), 4 collection tubes, waste beaker, and the Zyppy Plasmid Kit. 

1. Pipette 600 ul of grown E.coli broth into each ephindorph tubes (set #1)

    ->  Centrifuge tubes for 1 minute at 13.3 speed until pellet forms at the bottom corner

2. Dispose for supernatant in waste beaker but keep the pellet.

3. Pipette another 600 ul of E.coli broth into the tubes with 100 ul of lysis buffer and 350 ul of neutralizing buffer and invert until all solution forms a yellow solution with precipitation. 

    -> Centrifuge tubes for 2-5 minutes at 13.3 speed until there are little to no precipitation in the yellow solution. 

4. Pipette about 900 ul of supernatant into 4 columns in the collection tubes 

    -> Centrifuge tubes for 1 minute at 13.3 speed until supernatant is in the collection tube below. 

5. Dispose of supernatant from collection tube 

6. Pipette 200 ul of Endowash Buffer into each columns

    -> Centrifuge tubes for 30 seconds at 13.3 speed

7. Dispose of supernatant from collection tube

8. Pipette 400 ul of Zyppy Wash Buffer into each columns

    -> Centrifuge tubes for 1 minute at 13.3 speed

9. Dispose of the collection tube and supernatant and transfer columns into a new set of ephindorph tubes (set #2) 

10. Pipette 30 ul of Elusion Buffer into each columns 

11. Wait 1 minute and then centrifuge tubes for 1 minute at 13.3 speed 

    -> Dispose columns but keep the supernatant in the ephindorph tubes 

Results

Before we store away the tubes in the -20 C freezer, we would have to measure the cell density. To do this, we selected dsDNA on the nanodrop and used 2 ul of our Elusion Buffer as our blank. We loaded 2 ul of each solutions onto the nanodrop. In order to ensure that we have good cell densities, we want the ug/ml to be around 40-50 while our A260/A280 and A260/A230 to be around 1.8-2.0. 

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