Week 13 (4/1-4/5): Repeating transformation with pwTCas9 plasmid and D.caeni

Introduction

In lab this week, we repeated a transformation protocol with the pwtCas9 plasmid and D. caeni hoping that it can give us good results on plates to do plasmid confirmation. The beginning of the week comprise of growing plasmid culture in 20 mL of LB broth with 50 ug/ml of ampicillin. We also prepared TGY agar plates with 2ug/ml of tetracycline to observe our transformed cell growth. We hope that there will be transformed cells grown on the plates as the pwtCas9 plasmid was proven to be resistant to tetracycline. 

Plasmid Extraction Procedure

Below is a list comprising of how we performed plasmid extractions with pwtCas9. 

Solutions       # of Cell Packing        Resuspension        Elusion Buffer Amt

PC1                        1                                No                            10 ul 

PC2                        1                                No                            20 ul 

PC3                        1                                No                            30 ul 

PC1.                       1                                No                            10 ul 

PC2.                       1                                No                            20 ul 

PC3.                       1                                No                            30 ul 


Plasmid Cell Density 

Solutions       ng/ul        A260/A280        A260/A230

PC1                71.4            1.83                        0.40

PC2                33.7            1.80                        0.50

PC3                42.1            1.78                        0.60

PC1.               62.6            1.91                        0.75

PC2.              50.0             1.83                        0.45

PC3.              22.0             1.67                        0.76


Transformation Procedure

Since our ng/ul for each solutions were quite low, we did not have to dilute any of these in our transformation. We made 8 total centrifuge tubes for the transformation. Below shows the transformation protocol we followed. 

1. We thawed out our D. caeni competent cells on ice 

2. We then aliquot 100 ul of our competent cells into 6 centrifuge tubes. 

3. In the same 6 centrifuge tubes, we also put 10 ul of our plasmid solutions into each. 

4. After that, we placed the 6 microcentrifuge tubes comprising of our competent cells and plasmid extraction solution on ice for 15 minutes. This exposes the competent cells to heat shock which opens up the cells, allowing the plasmid in. 

5. After the 15 minutes, we incubated the tubes at 28 C for 45 minutes, remembering to agitate them every 15 minutes by inverting the tubes. 

6. While the microcentrifuge tubes are being incubated, we prepared 8 centrifuge tubes with TGY broth. 7 tubes will consist of 4 mL of TGY broth along with 2 ug/ml of tetracycline while 1 of the tubes will consist of only 4 mL of TGY broth. 

7. After the microcentrifuge tubes are incubated, they were transferred to the centrifuge tubes containing TGY broth and tetracycline. Only 6 of the centrifuge tubes contains 6 of the plasmid and competent cell solutions while 1 has our D. caeni control with TGY broth and tetracycline (this will be our negative control as we do not expect any growth since D. caeni is quite sensitive to tetracycline) and our last centrifuge tube will comprise of just D. caeni control with TGY broth and no tetracycline (this will be our positive control as we do expect growth from D. caeni) 

8. These tubes will be incubated together for at least 16 hours in the 30 C shaking incubator. 

9. After 16 hours, we plated 7 of these solutions onto the tetracycline and TGY agar plates that we have prepared and the positive D. caeni on normal TGY agar plates. 

Results

Unfortunately, after a total of 4-5 days, there was no growth at all on the plates consisting of the competent cells and plasmids solutions or the negative plate but only growth on the positive plate. We expected this to happen in a way because our plasmid solutions density was quite low (>10 ng/ul). Perhaps that our plasmids were low and that there were no growth on our plates, we conclude that no cells were able to be proven transformed this week. 

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