Bryan Hernandez/20.109/Lab notebook/Module 3/Day 4

--Bryanh 16:57, 13 April 2007 (EDT)
Purpose: To check if our SPT3 Knock-out worked via a Colony PCR analysis. Spot test candidate colonies for changes in physiology against the FY2068. Measure and compare RNA concentrations between our mutant strain and FY2068.

Protocols

Part 1: Agarose gel of PCR products

You will share an agarose gel with one other group.

1. Retrieve your PCR samples from the teaching faculty.
2. Move 10 ul of each sample to a labeled eppendorf tube.
3. Add 2 ul of loading dye to each of the eppendorf tubes.
4. Load these aliquots onto a 1% Agarose Gel (1xTAE), according to the following table.

Lane	Sample	Volume to load
1	100 bp Marker	5 ul
2	GROUP 1: PCR product/FY2068 template	~12ul
3	GROUP 1: PCR product/candidate A	~12ul
4	GROUP 1: PCR product/candidate B	~12 ul
5	GROUP 1: PCR product/candidate C	~12 ul
6	100 bp Marker	5 ul
7	GROUP 2: PCR product/FY2068 template	~12ul
8	GROUP 2: PCR product/candidate A	~12ul
9	GROUP 2: PCR product/candidate B	~12 ul
10	GROUP 2: PCR product/candidate C	~12 ul

The gel will run at 125V for approximately 45 minutes, and one of the teaching faculty will photograph it for you.

The results of the gel were dismal. The negative control showed only primer bands, which is a positive result, however the two candidate lanes had no bands, neither from primers or reaction products. This is hard to interpret precisely but the most likely explanation would be a systematic error in the PCR protocol or that our knock-in simply failed.

Part 2: Spot test candidates for phenotypes

1. Decide on the types of media you will examine and any growth conditions (temperature etc). Retrieve these plates and label them with the date, your team color and the strains you are examining.
   • we decided to make two comparisons against the FY2068:
     • temperature (both 30C and 37C)
     • drug resistance (rapamycin). the literature shows that knocking out SPT3 tends to lead to an increase drug resistance to rapamycin, which is why we are doing this experiment.
2. Vortex the strains that you innoculated last time: FY2068, candidate A, candidate B, and candidate C.
3. Next move 100 ul to the first well of a 96 well dish: FY2068 to position A1, candidate A to B1, candidate B to C1.
4. Add 90 ul of sterile water to the wells A2-A6, B2-B6, C2-C6.
5. Using your P20 set to 10 ul, pipet the yeast in well A1 up and down to mix them then move 10 ul of the yeast into the water that is in A2. Repeat, moving 10 ul of the dilution from A2 to A3 then 10 ul from A3 to A4 all the way out to A6.
6. Repeat the serial dilution series for the yeast in wells B1, C1.
7. Lay your labelled petri dishes open on the bench top.
8. Place 4 pipet tips on the multichanel pipetman and spot 3 ul from column 1 onto the leftmost side of each petri dish. You can use the same pipet tips for all your plates but be sure that the tips are properly filling each time. Sometimes liquid can accumulate in them to give errors in measurement.
9. Change pipet tips and then spot 3 ul from column 2 next to the spots you just placed from column 1. Spot all the petri dishes in this same way.
10. Repeat for columns 3-6.
11. Carefully replace the covers on the petri dishes but do not move them from your bench until all the spots have dried.
12. Wrap them in your colored tape and place them in the incubators.

Part 3: Isolate RNA

Once you know which, if any of your candidates have the SAGA-subunit deletion, proceed to isolate RNA from that strain and from the parent strain, FY2068. unfortunately, from our PCR results, it seemed that none of our knock-outs worked. as such, the results of the spot test might be meaningless if in fact the knock-out was unsuccessful.

Measure cell number

Microarray analysis allows us to compare the population of RNA from two samples with an identical number of cells, namely 2 x 10^7. Begin this part of today’s lab by measuring the density of your overnight cultures then converting this spectrophotometric measurement into cell number.

1. Make a 1:10 dilution (100 μl into 900 μl water) of each culture.
2. Use 0.5 ml to measure the optical density at 600 nm for each dilution, using water to blank the spectrophotometer.

	OD600nm	Cells/ml (using 5 OD600~108 cells/ml)	culture cells/ml	volume (ml) for 2E7 cells
FY2068	.5346	1.07E7	1.07E8	.187
SAGA mutant B	.8602	1.72E7	1.72E8	.116

Spheroplast

1. Collect 2 x 10^7 cells of each strain (on the order of 200 ul if the strains are both densely grown) in eppendorf tubes
2. Harvest by spinning the tubes in a microfuge, full speed, 1 minute.
3. Aspirate to remove all the supernatant.
4. Add 1 ml of supplemented Y1, resuspend the pellet, and incubate at 30° taped to the roller drum rolling at speed 4 for 15 minutes. Y1 is supplemented with zymolyase, an enzyme that will break down the yeast cell wall. yet another fatal accident occured here. after retrieving the samples from the incubator some of the sample spilled out of the eppendorf which voids the experiment because now there arent comparable quantities of RNA in both parent and experimental samples.damn
5. Microfuge your samples at 1.8 rpm = 300 rcf for 5 minutes.
6. Aspirate to remove all the supernatant from each sample. You can spin 1 minute more at 1.8 rpm to spin the last bits of liquid off the walls of the tubes and then aspirate or use a pipetman to remove.

RNA prep

1. Add 350 ul RLT+BME to each pellet. Pipet up and down to resuspend then vortex on minute to lyse spheroplasts. You can spin the samples at this point for 2 minutes if there is any insoluble material.
2. Add one volume of 70% EtOH. Pipet up and down immediately upon addition and apply sample to the spin column and collection tube that you can collect from the teaching faculty.
3. Microfuge 30 seconds at full speed.
4. Discard the flow-through into the sink or a conical tube that you set up on your bench for collecting waste. Save and re-use the collection tube for the next step.
5. Wash the column with 700 ul RW1. Microfuge 30 seconds at full speed.
6. Discard the flow-through and the collection tube. Get a new collection tube from the teaching faculty.
7. Wash the column with 500 ul RPE. Microfuge 30 seconds at full speed.
8. Discard the flow-through but re-use the collection tube.
9. Microfuge the column for 1 minute full speed to dry it.
10. Move the column to an RNase-free eppendorf tube with the cap cut off.
11. Elute the RNA from the column by adding 30 ul of RNase-free water directly to the membrane.
12. Allow the water to remain on the membrane for one minute, then microfuge, full speed for 1 minute.
13. Repeat with a second 30 ul aliquot of water, so each sample will yield approximately 60 ul of RNA.

Measure RNA concentration

1. Measure the concentration of your RNA sample by adding 5 μl to 495 μl sterile water. Use your P1000 to transfer the dilution to a quartz cuvette and measure the absorbance at 260 nm. Water in one of the optically paired cuvettes should be used to blank the spectrophotometer, but if another group has done this already, it does not have to be repeated.
   • A few things to be aware of when using quartz cuvettes:
     • They are very expensive.
     • The lab has only one set.
     • When you are done using the cuvette, you should carefully clean it by shaking out the contents into the sink and rinsing it two times with water. Quartz cuvettes get most of their chips and cracks when someone is shaking out the contents since it is so easy for the cuvette to slip from wet fingers or be hit against the sink. Don’t let this happen to you.
2. To determine the concentration of RNA in your sample, use the fact that 40 μg/ml of RNA will give a reading of 1 A260.

Because of our spill accident before the numbers shown below are taken from the green group because they were also doing the SPT3 knock-out.

RNA Sample	A260	Conc of dilute RNA	Conc of undiluted RNA
FY2068	.0236	.944ug/ml	94.4ug/ml
SAGA mutant	.0742	2.968ug/ml	296.8ug/ml

summary/interpretation

we got a lot of results today:

colony PCR: this, as explained before, was a negative result. no bands were found in the experimental lanes suggesting that the knock-out failed or there was a systematic error in our PCR procedure.
spotting: i dont expect to see very meaningful results here because the strains used were negative on the PCR analysis. The spot test might have showed interesting results with regards to the differences in growth on rapamycin plates since there is literature describing the increased fitness on rapamycin plates for SPT3 knock-out strains. we also expect to see an decreased fitness level on regular YPD. we will be able to say what phenotypical differences exits when we see our plates on friday.
RNA measurements: The SPT3 knock-out strain had a higher RNA concentration than the FY2068 strain. So knowing that "SPT3 interacts with Spt15p to activate transcription of some RNA polymerase II-dependent genes and also functions to inhibit transcription at some promoters," the results that we've seen today seem fairly consistent. by knocking out a protein that is involved in transcription regulation would most naturally lead to higher RNA levels as we have seen. there is some contradiction in the induction of some RNA polymerase II-dependent genes however it's hard to say which effect would be dominate. Judging by the results seen today, I would say that the stopping the gene inhibition dominates the stopping of gene induction.