Week 15 (4/15-4/19): Attempting confirmation cells on our existing transformed cells

 Introduction

Unfortunately since we could not successfully transformed pwtCas9 with D. caeni in Week 13, we will be using existing transformed cells that we have successfully gotten in the other previous experiments to attempt confirmation cells. There are two ways we can perform confirmation; PCR and Plasmid Prep. PCR is essentially the reading of genomic sequences of the cells and since we know that these transformed cells were successfully resistant to tetracycline, we want to see the resistant sequence gene once we load the PCR into a gel. Similarly, plasmid prep allows us to perform plasmid extraction on our transformed cell and loading them into a gel to see if it has the pwtCas9 plasmid within the cells. 


Procedure

For this confirmation, we used 4 different concentrations of the transformed cells and two controls:

- 135 ng pwtCas9 

- 550 ng pwtCas9 

- 783 ng pwtCas9 

- 803 ng pwtCas9 

- (+) D. caeni (no antibiotics)

- (-) D. caeni (with 2 ug/ml of tetracycline) 

All except positive D. caeni grown in a 15 ml centrifuge tube containing 3 ml of TGY broth and 2 ug/ml of tetracycline. Positive D. caeni was grown with only the 3 ml TGY broth. 

After almost 3-4 days, there was little to no growth in any of these tubes which can be concerning. We decided to only do PCR runs on the 783 ng pwtCas9 tube as it has the most growth out of all tubes. Below are our steps to perform PCR. 

1. We transferred 100 ul of our 783 ng pwtCas9 culture into a PCR tube.

2. We leave the PCR tube in the thermocycler at 95 C for 15 minutes. This essentially breaks down (denature)  the cell due to the high temperature. 

3. After that, we transferred 4 ul of the 783 ng pwtCas9 into a 36 ul mastermix consisting of our antibiotics primer (we did not have tetracycline but amp works fine as it is from E. coli), DNTP, polymerase, and buffer. 

4. 20 ul of this solution was transferred to a new PCR tube which goes into the PCR thermocycler procedure. Below shows a drawing of our timestamps and temperature. 








5. After this process, the PCR is then loaded on a gel in Week 17. 


Discussion

After almost 2 weeks of growing the transformed cells in the centrifuge tubes, there was still little to no growth. We theorize that this concerning event may have been that the cells might be dead or it may have lost the plasmid that gives it the resistant to tetracycline. Dead cells cannot grow in antibiotics due to the lack of resistant gene or it cannot grow at all. Thus, by sometime next week, we want to retry growing them again both on new plates (as we want to put them in freezebacks) and in new broth. 

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