Week 12 3/25-3/31: Transformation of PW1 Cas 9 results and more transformation

 Introduction

    In lab this week, we observe our PW1 Cas9 transformation results from Week 12. To recap, we plated P1 C and P1. C (reloaded 600 ul of culture after first spin), P2 LBR and P2. LBR (adding LB broth and reverting), and P3 LB (only adding LB broth). We also redid transformation of both pRAD 1 and PW1 Cas9 again. Below is our nano-dropping and cell density of each reaction. 

Table 

- pRAD 1 

Reactions    ng/ul    A260/A280    A260/A230    Amt of Elusion Buffer Added

P1                83.8        1.86                 1.65                    10 ul 

P2                38.1        1.83                 1.89                    20 ul

P3                45.5        1.87                 1.70                    30 ul

P1.               78.6        1.84                 1.79                    10 ul

P2.               60.3        1.86                 1.87                    20 ul 

P3.               46.7        1.86                1.69                     30 ul 

We diluted the mass of each of these reactions/plasmid with TE buffer. In the end, we were able to transform 92 ng, 120 ng, 234 ng, 380 ng, 450 ng, 600 ng, and 830 ng with 4ml of TGY and competent cells. Each of these plasmids were incubated in 30 C shaking and then plated on 3ug/ml of chloramphenicol and TGY agar. These are in the 30 C incubator until Monday. 

- PW1 Cas9

Reactions    ng/ul    A260/A280    A260/A230    Amt of Elusion Buffer Added

PC1              79.3        1.84                   1.74                10 ul 

PC2              64.1        1.88                    2.09               20 ul 

PC3             66.8        1.86                    2.09                30 ul 

PC1.            85.2        1.84                    1.92                10 ul 

PC2.            80.2        1.85                    2.31                20 ul 

PC3.            56.8        1.87                    2.17                30 ul

We diluted the mass of each of these reactions/plasmid with TE buffer like pRAD 1. In the end, we transformed 77 ng, 112 ng, 255 ng, 320 ng, 441 ng, and 800 ng with 4 ml of TGY and competent cells. These plasmids were incubated in the 30 C shaking and then plated on 2 ug/ml of tetracyline and TGY agar. These are also in the 30 C incubator until Monday. 

The reason why our plasmids and reactions have low and high cell density is so we can have a wide range of transformation. This gives us a stronger evidence of our transformation. 

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