Week 9 3/4-3/10: Loading into gel, do competent cells, transformation, and repeat plasmid extractions

 Introduction

    This week in lab was busy and productive as we were to complete our transformation outline before spring break. In the beginning of the week, we loaded our extracted plasmid that we did in week 9 onto a gel and run it. At the same time, our d.caeni successfully grew in broth without any contamination so we were able to do competent cells. After running the gel and competent cells, we were able to perform the transformation procedure. 

Gel Procedure

    To load a gel, we must obtain the required materials: 6 ul of loading dye and 6 ul of the 1k base pair ladder (we know that pRad 1 has a 7kb ladder so we want to expect that on the gel). To make the gel we mixed .30g of GelRed agarose and 30ml of TAE. 

    After obtaining these materials, we began to mix together the solutions. Since we want to run 3 lanes we would required 5 ul of pRAD1, 5 ul of PCR water, 2 ul of loading dye and 6 ul of the ladder. 

Gel Results

    After running the gel for 30 minutes 75 volts mA, this is what the gel looked like:

Shown here, only 1 of the plasmid appeared with the ladder. This conclude that we might need to do another plasmid extraction. 

    








Plasmid Extraction Results 

    Since the plasmid we performed last week did not show any significance on the gel, we decided to do another set of plasmid extractions. We followed the same exact Zyppy Kit Protocol again and these were the values we received after: 

               ng/ul     A260/A280    A260/A230

AN 1      49.2            1.90                2.21

AN 2      73.1            1.78                1.23

AN 3      46.1            1.90                2.00

AN 4      89.3            1.71                1.00

We will plan to put these reactions into a gel after spring break 

Competent Cells Procedure

Thankfully, our D.caeni grew in time so we were able to make competent cells. In order to do this, we require TGY broth, 60% glycerol, DI water, 1M of CaCl2, d.caeni broth culture (OD value: 0.9-2.0), microcentrifuge tubes (4), microcentrifuge, and 2ml cryovials.

1. Pipette 1 ml of broth culture into each sterile microcentrifuge tube

2. Centrifuge for 1 minute using microcentrifuge at 12000 RPM 

    -> Dispose supernatant 

3. Resuspend pellet in the following mixture: 

    - 1290 ul of TGY Broth

    - 51.6 ul of 1M CaCl2 

    - 335.1 ul of DI Water

    - 323 ul of 60% glycerol 

4. Mix well and place the solutions into 2ml cryovials

5. Store in -80 C freezer 

Transformation Procedure

Since we have made our competent cells and had our plasmid ready, we decided to just perform the transformation either way even if the plasmids did not show up in the gel. To prep, we would need microcentrifuge tubes, the 30 C incubator, ice, 2 15ml centrifuge tubes, competent cells, TGY broth, and pRad 1 plasmid. 

1. Thaw Competent cells in cryovial on ice

2. Aliquot 100 ul of competent cells into a microcentrifuge tube

3. Add 1 ug or <10 ul of plasmid solution itno the microcentrifuge tube

4. Place on ice for 15 minutes

5. Place in an incubator at 30-32 C for 45 minutes, agitating every 15 minutes by inverting tubes

6. After transferring content into 15 ml centrifuge tubes with 4 ml of TGY broth, incubate in a shaking incubator at 30 C for 16 hours. 

7. After the 16 hours, plate the cells onto chloramphenicol TGY plates and let them sit for 4 days in the 30 C incubator. 

For this we only did two tubes, JT1 and AN1. We pipetted 50 ul of each after the 16 hours onto the plates. We also did 50 ul of plain d.caeni onto a plate as a negative control. 

Transformation Results


From what we are seeing after the 4 days, our negative control had no growth. Our JT1 has small pink colonies while our AN1 had no growth. 





Comments

Post a Comment

Popular posts from this blog

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

Image
  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 ...
Read more

2/12-2/18: Week 6 Tetracycline vs Caeni efflux pump experiment

Image
 Introduction In lab this week, my group and I did one more testing with Tetracycline and Caeni. However, based on the results last week, we decided to do the concentrations below 1 ug/ml. The concentrations are 0 ug/ml, 0.25 ug/ml, 0.5 ug/ml, and 0.75 ug/ml. We expect at least some growth between 0 ug/ml and 0.5 ug/ml.  Procedure  To prep, we made four 25ml flasks of normal TGY agar. The reason for this is because we want to ensure that each TGY plates we will be pouring is evenly 25ml. We then recieved our 2.5 ug/ml tetracycline stock from Chad. We also grew our caeni in a 10ml TGY flask.  To keep in mind, our OD value of caeni was 2.44 after 3-4 days of growth and was diluted to 0.97. 1. We label each of four 25ml flasks of TGY agar their desired tetracycline concentrations and also labeled four empty plates the corresponding concentrations to the flasks.                    Flask #1 and Plate #1: 0ug/ml...
Read more