Week 10 Lab JW

Goal: The goal of this lab was to observe the phenotypes of the yeast cells on ½ YPD media and purify DNA for sequencing.

 

April 22, 2014

Broth Cultures: From the ½ YPD plate, 2 red colonies, and 2 white colonies were isolated and inoculated into separate broth cultures. The broth cultures contained 2.5 mL of YPAD and 500 uL of sterile glucose at 20%. The tubes placed in shaker at 30*C overnight.

 Image

Figure 1: Showing C2 colonies growing on the 1/2 YPD plate

April 23, 2014

Performed Plasmid rescue: Mini-prep was performed as it was in experiment 2 on the 4 colonies. However when P1 buffer was added, so were 100 uL of glass beads. The beads help break down the cell membranes of the yeast cells when vortexed. Mini-prep protocol was followed the same as before with 45 uL elution volume.

Mini-prep DNA that was harvested was then used as a template for PCR. A total of 4 reactions were prepared as followed:

9 uL of Taq Master mix

1 uL of template DNA

1 uL of primer mix SEQ

7 uL of sterile water

The reaction was heated in the thermocycler as stated before, except the difference is 35 cycles instead of 30.

 

April 24, 2014

PCR reactions were purified using the NucleoSpin kit as previously described.

Week 11 Lab JW

Goal: To ensure that we have DNA for sequencing and to prepare the sequencing samples and send them out for analysis.

 

April 30, 2014

PCR amplification from the previous week was run on a 1% agarose gel. To ensure that the DNA showed up, 4 uL of DNA were loaded with 2 uL of dye.

bmb lab 11 fig

Figure 1: DNA from PCR reactions. On the top row two in from the right are my Red1 and Red2. On the bottom row two in the from the right are my White1 and White2.

 

All of my samples contained DNA and were prepared for sequencing. 2 uL of purified DNA was added to 8 uL of dH2O and was mixed with EDR259 sequencing primer (5 uL and diluted 10-fold) in PCR tubes. It was then packaged and sent to GeneWiz.

Week 9 Lab JW

Goal: To remove WT SUP35 plasmid, leaving behind a prion and mutated versions of SUP35.

 

April 14, 2014

Yeast collected from SD+A+T+U plates with 1 mL of water and spreader. It was then plated onto a 5-FOA plate. 5-FOA plate removes WT SUP35 plasmid allowing for only the mutated SUP35 to grow. Once all the cells had soaked in, they were put into an incubator at 37*C and grown for 3 days.

 

April 18, 2014

Again, the yeast were collect from the 5-FOA plates with a spreader and 1 mL of sterile water. A 1:100 dilution was made the same way as in previous experiments and the OD at 600 nm was taken with the spectrophotometer. The OD of the C2 culture was 1.045. In order to get the correct dilution a 1:1000 dilution was made. 2 uL of the C2 was added to 200 uL of sterile water. It was then spread on a ½ YPAD plate and placed to incubate at 30*C for 5 days.

Week 8 Lab JW

Goal: The goal of this lab was to collect transformants and mate yeast with prion positive yeast on selective plates.

 

April 9, 2014

Observation of the SC-leu plates: Amount of colonies on the low, high, and negative control were counted. There were approximately 50 colonies on the negative control, 3 colonies on the high, and 0 on the low.

 

Mating of YER258 and YER648: The cells were collected from the C2 high plates by spreading around 1 mL of sterile water. The concentration of YER648 and YER258 (WT yeast) were checked in the spectrophotometer. A 1:100 dilution was made with sterile water (10uL of yeast, 990 uL sterile water). The OD of YER258 was .125, while the OD of YER648 was .215. For mating, 250 uL of YER648 and 430 uL of YER258 needed to be added to the YPAD plate. Without touching or spreading the cells after they were poured onto the plate, they were incubated at 30*C for 24 hours.

 

***Used KB YER258 cells as the amount on my plate was not enough to collect and measure.

 

An SD+A+T+U plate was made simply by spreading them on a SD plate.

 

April 10, 2014

The freshly mated yeast were then collected with 1 mL of sterile water and spread on the SD+A+T+U plate. They were then placed in the incubator at 30*C for four days.

Week 7 Lab JW

Goal: The goal of this lab was to verify size of the 2-piece PCR reactions, prepare yeast for transformation, and to transform mutant PCR constructs and double digest pER154 backbone.

 

March 20, 2014

2-piece PCR reactions were purified using NucleoSpin kit as described previously. They were then run on gels for correct sizing.

bmb lab 7

Figure 1: Agarose gel run to check sizing of 2-piece PCR reaction. Lane 1: Ladder, Lane 8: Jillian’s C2 reaction

 

Recombination should occur between two linear DNA pieces creating a library of Sup35 mutants. All mutations should be in the 4th oligorepeat of the propagation domain.

 

March 25, 2014

Inoculated a culture of YER648 in YPAD broth and 20% glucose. Incubated the culture in shaker at 30°C. Solution had optical density of 0.33 at 600 nm.

 

April 2, 2014

10 uL of double-digested and purified pER154 was added to 5 uL of purified 2-piece PCR reaction in a microfuge tube. In a separate tube, 10 uL of the double-digest pER154 was mixed with TE buffer (40 uL) to be used as a negative control. Salmon sperm carrier DNA was then boiled for 5 minutes and put on ice. The yeast cells that were grown overnight were spun at 1.5k rmp for 5 minutes. 1 mL of TE buffer was then added after disposing of the supernatant. It was then transferred to a microfuge tube and spun at 13k rpm for 30 seconds. Supernatant was discarded and then the pellet was  re-suspended in 150 uL of TE buffer. Next 15 uL of the salmon sperm DNA was added to the re-suspended pellet. 50 uL of yeast cell and salmon sperm DNA was added to the 2-piece PCR and the negative control. Next it was vortexed and 150 uL of lithium acetate was added. 1 mL of PEG solution was added both tubes were vortexed. They then incubated at 42*C in a water bath for 15 minutes, followed then by centrifuging at 13k rpm for 45 seconds. The pellet was re-suspended in 200 uL of dH20.

Plating: 190 uL of re-suspended cells transformed with the recombined plasmid were plated on an SC-leu plate of 1x dilution and the rest was spread onto an SC-leu plate of 1/20x dilution. 1x dilution of cells from the negative control were spread onto a different SC-leu plate. They were then incubated at 30*C for 3 days and then moved to the refrigerator.

Week 6 Lab JW

Goal: The goal of this lab was to check that our DNA was amplified correct as well as to perform a “2-piece” PCR reaction to link our N2 and C2 products.

 

March 19, 2014

Gel check was performed. The PCR reactions from the previous experiment were checked using a 1% agarose gel.

bmb lab 6 c2

Figure 1: C2 PCR reaction. Lane 1: 500 bp ladder, Lane 8: Jillian’s reaction

bmb lab 6 n2

Figure 2: N2 PCR reaction. Lane 8: 500 bp ladder, Lane 7: Jillian’s reaction

As seen above, the reactions contained the DNA of the appropriate size. Original PCR ractions were purified using the NucleoSpin protocol that has been previously described (experiment 3). The final elution volume was 30 uL.

 

PCR set up: 2-piece PCR reaction contained: 30 uL Taq master mix, 1 uL of purified N2 DNA, 1 uL of purified C2 DNA, 2 uL of primer mix 2P (15 uL of EDR302 and 15 uL of EDR262), and 26 uL of dH20. The thermocycler protocol was then followed the same as in experiment 3 with the exception that there was a 2 minute extension at 72°C and 35 cycles were run instead of 30.

Week 5 Lab JW

Goal: The goal is to run the PCR reactions on gel, cut out the band and purify the DNA, as well as to run a second round of PCR reactions to generate mutant forms of Sup35.

 

March 12, 2014

1% Agarose Gels: The PCR reactions for the previous week were run on agarose gels with a 500 bp ladder.

 

Figure 1: N1 PCR reactions. Lane 1-Ladder, Lane 8- Jillian’s reaction

BMB Lab 5 N1

Figure 2: C1 PCR reactions. Lane 8-Ladder, Lane 7-Jillian’s reaction

BMB lab 5 c1

Based on expected size of N1 and C1 PCR reactions, the bands seem the appropriated size.

 

The bands with the N1 and C1 DNA were then cut out of the gel and placed into microcentrifuge tubes. The DNA was then purified using the NucleoSpin Gel and PRC Clean-up kit the exact same way as was done before (03/05), except in the last step, the DNA was eluted with 15 uL. The N1 and C1 purified DNA was diluted 1:20 and 2nd round PCR reactions were set up.

 

2nd Round PCR Reactions: The N1 and C1 purified DNA that was diluted 1:20 was used for the 2nd round PCR reactions. In the N2 reaction, 9 uL of Taq master, 1 uL N1 (purified and diluted) DNA, 1 uL primer mix N2, and 7 uL of dH20 was added. The same was done for the C2 reaction, but instead of the N2 primers (EDR257 and KSM2) being added, the C2 primers (KSM1 and EDR262) were added. The thermocycler was then run as previously described in experiment 3.

Week 4 Lab JW

Goal: The goal of this experiment was purify the restriction digest from the previous week and to then perform a second one using BamHI, as well as to use PCR to amplify N1 and C1 reactions.

 

March 5, 2014

Purifying Plasmid DNA: The plasmid that was digested in the previous week was purified using the NucleoSpin Gel and PCR Clean-up kit. 200 uL of Buffer NTI was added to the 100 uL sample and centrifuged at 11,000 x g. The column was then washed with 700 uL of Buffer NT3, and was centrifuged for 30 seconds. This step was repeated once more. The column was centrifuged again for 1 minute. DNA was eluted from the column with 45 uL of Buffer NE, rested for 5 minutes, and then centrifuged for 1 minute where the flow through was kept.

 

2nd digestion and Re-purifying: 10 uL of BamHI buffer, 1 uL 100x BSK, 45 uL dH2O and 2 uL BamHi were added to the purified DNA. The tubes then digested overnight at 37°C in a water bath. The next day (03/06), the digest was removed and purified following the same procedure as above. The tube was then stored in the freezer.

 

PCR-amplifications: Performed on pER243 which is a plasmid that contains the WT Sup35 protein. 9 uL Taq master mix, 1 uL 1/500x pER243, 1 ul primer mix N, and 7 uL dH2O were added to a PCR tube to form the N1 reaction. 9 uL master mix, 1 uL 1/500x pER243, 1 uL primer mix C, and 7 ul dH20 were combined into a second PCR tube to form the C1 reaction. The tubes were then placed in the thermocycler and run together at 95°C for 2 minutes, then at 95°C for 30 seconds, 54°C for 30 seconds, and 72°C for 1 minute. This was repeated 30x. They were then run at 72°C for 10 minutes and then at 4°C forever. These reactions will be run on 1% agarose gels and then purified.

Week 3 Lab JW

Goal: The goal of this lab was to isolated and purify DNA from last week’s transformation, to check for the presence of DNA and to perform restriction endonuclease digests of the plasmid.

February 25, 2014

Procedure:

Two 5-mL tubes of T-Soy Broth with Ampicillin were incoculted with separate colonies from the “high” plates from week 2. The were left to shake at 37° for 24 hours.

 

February 26, 2014

The cultures that were inoculated the night before were miniprepped. They were centrifuged at 13,000 rpm for 60 seconds. The pellets were re-suspended in 250 μL of Buffer P1. 250 μL of Buffer P2 was then added to lyse cells and was mixed by inversion. 350 μL of Buffer N3 was added to neutralize. The cells were centrifuged for 10 minutes to pellet. The supernatant was applied to at QIAprep spin column and centrifuged again for 1 minute, and then flow-through was discarded. The column was washed next with 0.75 mL of Buffer PE and centrifuged for 60 seconds and the flow-through was discarded. It was then centrifuged once more for 60 seconds to remove left over buffer. The DNA was then eluted by putting the spin column in a micro-centrifuge tube and adding 50 μL of Buffer EB. This set for 5 minutes and was then centrifuged for 60 seconds.

Agarose gels were made by dissolving .5 grams of agarose into 50 mL of TBE buffer and placing in the microwave. Once solid agarose was gone, 2 uL of gel red was added and it was poured into the proper tray. 4 uL of plasmid was mixed with 2 uL of the loading dye and run on the get for 45 minutes. The gels were then imaged

BMB Lab 3pic

Figure 1: Agarose gel image showing DNA presence. Lane 5 and 6 contain my DNA.

 

A restriction digest using HindIII was set up for the DNA that was revealed by the agarose gel. 50 uL of purified plasmid DNA, 10 uL of NEB2, 1 uL of 100x BSA (bovine serum albumin) solution, and 39 uL of dH20 were combined in a microfuge tube. The solutions were mixed and HindIII was added. The digest was put in a 37°C water bath for 24 hours. They were then moved to the freezer at -20°C.