This week marked the beginning of our next main project on Mimulus gutatus plant samples. For this lab we focused on DNA extraction. Prof Paul gave us the samples we collected in the field whenever we went to Mount Tamalpais with the class + Alec along with two other samples from other sites.
- The first step was to label 3 2mL tubes with our sample codes – which are shown below.
Tube Numbers / Sample Name / Sample ID Code
Tube 1: CATB- OY / OY01
Tube 2: MONO- 008 / OY02
Tube 3: DIRA- 001 / OY03
- Next, we added three sterile 3.2 mm stainless steel beads to each tube along with a small amount of leaf tissue to each tube, wiping off the tweezers between each sample. The steel beads were used to break up the leaf samples.
- The tubes were loaded onto a tube rack in the modified reciprocating saw rack and the rack was mounted to the saw. Prof Paul reciprocated the samples for about 40 seconds on speed setting 3.
- The tubes were then spun in the centrifuge to pull the plant dust from the lids for about 20 seconds.
- Next, we added 434 microliters of preheated grind buffer that was heated in the water bath.
- After adding the buffer we put our samples into the water bath that the grinder was in (65 degrees) for ten minutes, investing them every 3 minutes (3 times in the 10 min)
- 130 microliters of 2M pH4.7 potassium acetate was then added. The tubes were inverted several times and incubated on ice for 5 minutes
- Samples were then centrifuged on max force for 20 minutes. Centrifuge was balanced
- Next, we labeled new 1.5 mL tubes with the sample codes. Supernatant was transferred to the new tubes, while avoiding transfer of any precipitant that was at the bottom.
- We then added 1.5 volumes of binding bugger. (600 microliters)
- After this we put 650 microliters of the mixture to Epoch spin column tubes and centrifuged for 10 minutes at 15,000 rpm in a centrifuge, then discarded the flow-through in a hazardous waste container (Erlenmeyer flask in our case)
- Then we did this again with the remaining volume.
- Next we washed the DNA found to the silica membrane by adding 500 mL of 7-% EtOH to the Columbus and centrifuged it at 15,000 rpm until all the liquid passed to the collection tube (8min) and discarded the flow through
- Then we did this again ^
- Following that, we centrifuged the columns at 15,000 rpm for another 5 min to remove any residual ethanol
- Collection tubes were discarded and we put the columns in sterile 1.5 mL micro centrifuge tubes which were labeled with sample codes and the date
- NOTE: I put the columns in the wrong tube (the one containing our old DNA) and put the 100 microliters of preheated pure sterile H20 in those tubes… After noticing my mistake Prof Paul said to put it in the new sterile tubes and we ran 70 microliters of the pure sterile water through that into the tubes with the date on them and let that stand for 5 min
- These new tubes were centrifuged for 2 min to elute the DNA
- The tubes that I accidentally used first were labeled with a STAR
- Kept all the tubes in case