Sarah’s notebook entries are here: http://sspatty.wordpress.com/
Yura’s notebook entries are here: http://ysimbmblab.blogspot.com/
This week the sequences of DNA retrieved from the plasmid rescue were received an analyzed. The mutated sequences for the third repeats of the yeast colonies from row E are listed below. Sequences from colonies E1 and E3 are not listed as there were no mutations in these sequences.
E2 W Q F C Q A S F S A G
E4 R R V F G G G R A C R
E5 R P D T G Y H Q Q Y N
E6 R P R P N V I S F G A
The purpose of this week’s experiment was to run the PCR products from Week 9 through gel electrophoresis, and to determine whether the bands were of the correct size so that they can be used for sequencing. Image below.
Figure 1. Bands of PCR products. From left: lane 1-ladder, lane 2-8 (G1-G6), lane 9-skipped, lane 10-15 (Yura Sim).
Each reaction (4 µL) was placed into the well of agarose gel (1%), along with a ladder (5 µL). Given my bands were of the correct size, they were cut out and purified using the Nucleospin Gel and PCR Clean-up protocol (see week 3, correction: NE buffer (30 µL). Then mixtures for sequencing were set up by mixing purified sample (10 µL) with EDR259 primer (5 µL). Once mixtures were set, they were sent for sequencing.
The PCR products from Week 9 were run on a 1% agarose gel (4μL for each reaction, 5μL 500bp ladder) and visualized (Figure 1).
Figure 1: DNA gel of PCR products. From left to right: ladder, blank, F1, F2, F3, F4, F5, F6, blank, H1, H2, H3, H4, H5, H6.
The reaction mixtures were purified as described in Week 2 and eluted into NE buffer (30μL). The samples were then loaded into strip tubes for sequencing as follows: purified DNA (10μL) and 1:5 diluted EDR259 primers (5μL).
The F1-F6 colonies that were incubated in YPAD broth were subjected to a Miniprep plasmid rescue as follows. After pelleting the yeast cells, they were resuspended in Buffer P1 (250μL) and an equal volume of glass beads, after which the suspension was vortexed for 10 minutes. Buffer P2 (250μL) was added and the tubes were inverted 6 times. Buffer N3 (350μL) was added and the resulting mixture was inverted 6 times. The tubes were centrifuged for 10 minutes at 13,000 rpm. The supernatant was added to a QIAprep spin column and this was centrifuged for 30 seconds. After discarding the flow-through, Buffer PB (500μL) was added and the mixture centrifuged for 30 seconds. The flow-through was again discarded, and Buffer PE (750μL) was added and the mixture centrifuged for 30 seconds. The flow-through was discarded and the columns centrifuged dry for 60 seconds. Buffer EB (30μL) was added to the columns, which were incubated for 5 minutes and centrifuged for 60 seconds into clean 1.5mL centrifuge tubes to elute the plasmid DNA.
The purified plasmid DNA was added to strip tubes for PCR. One individual tube on the strip was used for the DNA of each colony. Pre-prepared primer SEQ consisted of primers EDR259 (2μL), primer EDR262 (2μL), and water (8μL). Each tube consisted of Taq master mix (9μL), plasmid DNA (1μL), SEQ primer mix (1μL), and DI water (7μL). The tubes were placed in the thermocycler and set at the same cycle as described in Week 4.
Previously, we practiced stamping colonies from a 96-well plate to a 1/2 YPD plates.
The protocol from Week 6 was repeated, and colonies were stamped onto a YPAD plate in addition to the 1/2 YPD plate. Both plates were incubated at 30°C. On the following Monday, the YPAD plates were wrapped in parafilm and stored at 4°C.
The phenotypes of colonies in F1-F6 were: white, white, white, red, red, and red, respectively. These colonies were picked and incubated in YPAD broth (3mL) cultures in the shaking incubator overnight.
Last week, we plated yeast cells on SC-Leu plates to select for colonies that consisted of cells that had been successfully transformed with our 2-piece PCR product.
Likely due to PCR-related problems, no usable colonies grew on the SC-Leu plates. From this point on, we will be using clones with mutations in the 3rd repeat of the oligopeptide repeat domain. Because we were still waiting for the clones to arrive from Colorado, we practiced stamping colonies. Colonies were stamped from a 96-well plate to a 1/2 YPD plate and incubated at 30°C.
The plates were removed from the incubator and left at room temperature for the weekend.
After each member of the class was assigned a row (I have row F), we observed the phenotypes of our stamped colonies.
This results of the PCR reaction from the preceding week were run through gel electrophoresis using the protocol from week 2. 4µl of each reaction was run in each well with 5µl of a 500bp ladder. Seen below is the gel.
Figure 1: Visualization of PCR product
Once it was determined that bands of the correct size were present each reaction was purified using the Nucleospin protocol from week 3 with the elution done in 30µl NE buffer. Following this 10µl of the purified sample was combined with 5µl of a 1:5 dilution of the EDR259 primer in strip tubes. These were sent to Genewiz to be sequenced.
The purpose of this week’s experiment was to perform a Miniprep protocol for plasmid rescue of yeast plasmids, and to set up PCR reactions using the miniprep plasmids as the template.
Tubes were labeled (G1,G2,G3…etc). In a microcentrifuge tube (1.5 mL) our cells were (1.5 mL) were centrifuged in order to pellet them. This is essential for the forthcoming vortexing step. The pelleted bacterial cells were resuspended in Bufer P1 (250 μL). Glass beads of the same volume were added and mixture was vortexed (10 min). Buffer P2 was then added (250 μL) and inverted (4-6 times). Buffer N3 was then added (350 μL) and mixed immediately by inversion. The solution was centrifuged (10 min, 13000 rpm). The supernatant was then pipetted to a QIAprep spin column. Again the solution was centrifuged (30 s). The flow through was discarded. The QIAprep spin column was then washed by adding Buffer PB (0.5 mL) and centrifuged (30 s). Again, any flow through was discarded. The QIAprep spin column was washed again by adding Buffer PE (0.75 mL) and centrifuged (30 s). Flow through was discarded. The solution was centrifuged once more to remove any residual wash buffer. In order to elute the DNA, the column was placed in a clean microcentrifuge tube (1.5 mL) and Buffer EB (30 μL) was added. The solution was allowed to stand for 5 min, followed by centrifugation (1 min).
The PCR reactions were then set up using these purified samples as the templates. Six PCR reaction tubes were set up, representing the six individual clones. The mixture for each consisted of respective purified DNA samples (1 μL), Taq master mix (9 μL), primer mix SEQ (1 μL), and sterile dH2O (7 μL). The tubes were then placed in the thermocycler with the following cycle:
95 C-2 min, (95 C-30 s, 54 C-30 s, 72 C-1 min)X35, 72 C-10 min, and 4 C (till removed).
Note: Clones to be used for the remainder of the semester arrived. The stamping process that was practiced last week was utilized this week. The purpose of this weeks experiment was to stamp MY colonies (Row G) onto a fresh 1/2 YPD plate.
Using wooden sticks, colonies were picked from my row and placed into a 96-well plate (already contained sterile dH2O (100 μL)). With the stamper, colonies were stamped onto a 1/2 YPD plate and a fresh sc-leu plate (YPAD). Both plates were then placed in the incubator (30 C). The colonies on the 1/2 YPD plate were allowed to grow for 2 days. The plate was then taken out of the incubator and placed on the bench to allow for ripening. The phenotypes were determined to be : white, white, white, red, red, red. Clones were then labeled 1-6.
6 YPAD cultures were then set up. This was done by placing in the culture tube (3 mL) a “glob” of yeast from the respective clone number in the row (ex. clone 1=culture tube 1). This was repeated for the remaining 5 clones. The cultures were then grown with shaking in the shaking incubator overnight.