Lab Post #5 (9/12/16)

There are multiple ways to transform yeast, but today we will be working by the Lithium Acetate Model.

We started with the yeast that was inoculated last lab day. The eppendorf tube was centrifuged at 6000 rpm for 5 minutes in order to harvest the cells. Using a pipette, we removed all of the supernatant in the tube. The pellet was left over at the bottom and so we cleaned it by vortexing it with 1mL of sterile H2O (ddH2O). Afterwards, all of the H2O had to be removed using a pipette. We were not able to do it all at once, and so we centrifuged again for 3 minutes at 6000 rpm.

Afterwards, 100mM of Lithium Acetate (LiAc) solution was added to the pellet. We were provided with 1M of the solution and so we diluted it by mixing 20mL of LiAc with 180mL of ddH2O. Our calculation:

C1V1=C2V2 -> V1=(C2V2)/C1

C2=100mM H2O     V2=200uL LiAc     C1=1000mM H20

V1 = (100mM)(200uL)/1000mM = 20uL LiAc

180 uL H20 + 20 uL LiAc = Total Volume 200uL for 100mM LiAc

 

The solution and pellet were vortexed for a few seconds before being incubated at 30 degrees Celsius for 10 minutes. The tube was centrifuged at 6000 rpm for 30 seconds so that we could remove the supernatant. The pellet was then resuspended in 36uL of LiAc, 25 uL of Salmon sperm, 50 uL of plasmid DNA and water (6 uL), 240 uL of 50% PEG (w/v), and 30 uL DMSO. We used 6uL of the plasmid DNA last class for the gel electrophoresis and so we replaced that amount with 6uL of ddH2O. Then eppendorf tube was vortexted and then incubated at 30 degrees Celsius for 30 minutes and then the cells were given heat shock by being incubated at 42 degrees Celsius for 15 minutes.

*Heat shock is for 15 minutes although the lab protocol states 20 minutes.

After heat shock, cells were pelleted for 5.5 minutes at 8600 rpm to allow for the supernatant to be removed.

Lastly, we resuspended the pellet in yeast extract peptone dextrose (YPD).

*We used YPD instead of ddH2O as stated in the protocol because it is a growth medium.

We resuspended by slowly pipetting up and down with the 200uL of YPD. We then plated the solution in an agar plate along with 8 glass beads and shook the plate lightly in order to spread the yeast.

 

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