PROTOCOLS FOR EXPERIMENT 4 (Labs 7, 8 and 9)
Lab 7: RNA isolation and Pictures of plants
Goal: to study light regulated gene expression using mutants and RT-PCR.
You will be extracting RNA from 4 samples:
1. wild-type grown in the light
2. wild-type grown in the dark
3. det1 grown in the light
4. det1 grown in the dark
Pictures of plants:
1.) Pick seedlings from the four plates.
2.) Arrange seedlings in a petri dish.
3.) Using the digital camera provided take a digital image of the seedlings.
RNA isolation:
1.) Pick your seedlings (approximately 40-50 mg of tissue).
2.) Place seedlings in a leak-proof 1.5 ml homogenization tube that has been immersed in liquid nitrogen. Add some liquid nitrogen.
3.) Let sit for 30 seconds and then briefly crush the tissue with the homogenizer pestle that you have dipped in liquid nitrogen.
4.) Add 500 ul of Trizol reagent. Trizol is a very HARMFUL substance: BE CAREFUL.
5.) Homogenize the tissue for 1 minute.
6.) Let stand for 5 minutes at room temperature.
7.) Add 100 ul of chloroform and shake the tube for 15 seconds. Be careful that the lid is closed tightly.
8.) Let stand for 2-3 minutes at room temperature.
9.) Centrifuge at top speed in a Microcentrifuge for 10 minutes.
10.) Transfer the colourless upper phase to a fresh labeled 1.5 ml microcentrifuge tube.
11.) Add 250 ul of isopropanol to the transferred upper phase.
12.) Let stand at room temperature for 10 minutes.
13.) Place on ice for 3 minutes.
14.) Centrifuge for 10 minutes at top speed in a Microcentrifuge .
15.) Remove and discard the supernatant and retain the gelatinous pellet.
16.) To wash the pellet: Add 500 ul of 75% Ethanol and vortex to resuspend the pellet.
17.) Place on ice for 3 minutes.
18.) Centrifuge for 5 minutes at top speed in a Microcentrifuge .
19.) Remove and discard the supernatant.
20.) Let air dry for 10 minutes.
21.) Add 25 ul of DEPC treated water.
22.) Incubate at 45°C for 10 minutes.
23.) Take a 5 ul sample of each total RNA prep, add 5 ul of 5X sample buffer, and run it on an agarose gel.
24.) Take a picture of the gel. REMEMBER TO SAVE YOU IMAGE AS A .TIF FILE (RAW DATA). AND MAKE SURE YOU HAVE SAVED IT TO THE RIGHT FOLDER.
25.) Store samples of total RNA in the freezer.
Lab 8: RT-PCR
Step 1: Reverse transcription of mRNA.
-For Step 1 you need the following: Reverse
transcriptase, dNTPs, buffer, DTT and poly(dT)12-18 primer.
-Set up the following 4 reactions in 1.5 ml microcentrifuge
tubes:
| Component | 1 | 2 | 3 | 4 |
| wild-type total RNA; light | 5 ul | - | - | - |
| det1 total RNA; light | - | 5 ul | - | - |
| wild-type total RNA ; dark | - | - | 5 ul | - |
| det1 total RNA; dark | - | - | - | 5 ul |
| water | 4 ul | 4 ul | 4 ul | 4 ul |
| dNTPs | 2 ul | 2 ul | 2 ul | 2 ul |
| oligo(dT)12-18 primer | 2 ul | 2 ul | 2 ul | 2 ul |
| Total volume | 13 ul | 13 ul | 13 ul | 13 ul |
To melt secondary structure: incubate the tubes at 70°C for 5 minutes, and chill on ice. Spin the tubes in a microcentrifuge for 20 seconds.
To each of the four reactions add:
| Component | Volume |
| 5X 1st strand buffer | 4 ul |
| 0.1 DTT | 2 ul |
| M-MLV reverse transtriptase | 1 ul |
| Final volume | 20 ul |
Mix gently by pipetting up and down.
Incubate in a 37 °C waterbath for 50 minutes.
To stop the reaction: incubate the reactions at 70°C for 15 minutes, and place tubes on ice.
At this point you will have made the first stand of a cDNA copy of the mRNA population. The next step is to PCR amplify a specific template from this cDNA copy of an mRNA population.
Step 2: Multiplex PCR amplification.
Set up the following 8 reactions in 200 ul PCR tubes. Remember that you have to make master mixes (it saves time and the pipettes cannot dispense a volume less than 0.5 ul).
| Component | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| wild-type; light cDNA (1) | 5ul | - | - | - | 5ul | - | - | - |
| det1;light cDNA (2) | - | 5ul | - | - | - | 5ul | - | - |
| wild-type; dark cDNA (3) | - | - | 5ul | - | - | - | 5ul | - |
| det1; dark cDNA (4) | - | - | - | 5ul | - | - | - | 5ul |
| Primer UBIQUITIN FOR | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul |
| Primer UBIQUITIN REV | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul |
| Primer CLAB FOR | 1ul | 1ul | 1ul | 1ul | - | - | - | - |
| Primer CLAB REV | 1ul | 1ul | 1ul | 1ul | - | - | - | - |
| Primer CHAB FOR | - | - | - | - | 1ul | 1ul | 1ul | 1ul |
| Primer CHAB REV | - | - | - | - | 1ul | 1ul | 1ul | 1ul |
| 10X PCR buffer | 5ul | 5ul | 5ul | 5ul | 5ul | 5ul | 5ul | 5ul |
| 50mM MgCl2 | 3.0ul | 3.0ul | 3.0ul | 3.0ul | 3.0 ul | 3.0 ul | 3.0 ul | 3.0 ul |
| 5mM dNTP | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul | 1ul |
| water | 31.75ul | 31.75ul | 31.75ul | 31.75ul | 31.75ul | 31.75ul | 31.75ul | 31.75ul |
| Taq DNA Polymerase | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul | 0.25ul |
| total | 50ul | 50ul | 50ul | 50ul | 50ul | 50ul | 50ul | 50ul |
Give to the TA to start the PCR reaction.
Store PCR reactions in the freezer.
Lab 9: Polyacrylamide gel electrophoresis of plant-derived RT-PCR samples
1. Set up an 6% polyacrylamide gel. Polyacrylamide gel electrophoresis
2. Add 13 ul of 5X sample loading buffer to each RT-PCR reaction and load 20 ul on gel.
3. Lane 1 should contain the 100 bp ladder and lanes 2-9 should contain the 8 RT-PCR reactions.
4. Run the gel at 100 volts.
REMEMBER TO SAVE YOU IMAGE AS A .TIF FILE (RAW DATA). AND MAKE SURE YOU HAVE SAVED IT TO THE RIGHT FOLDER.