We conducted a double digest restriction associated DNA study on Mimulus gutattus. The first step was collecting samples, which was done on two field trips (Lab 02 and Lab 05)
Next, we extracted DNA from collected samples as well as previously collected samples by Our Boy Alec. After, we double digested our DNA using two restriction enzymes, cutting the DNA into many pieces. Then, we ligated unique DNA barcodes onto each individual. Our next step was using PCR to add a second unique barcode and to test if our library construction was successful. The library construction was successful. After the test PCR, a larger reaction was conducted that was identical to the aforementioned process. This was the last step performed as a class and in a perfect world, the next steps would start with size selection. Size selection selects DNA of specific sizes (duh), but specifically we would be targeting 400-600 bp fragments. This is the typical size for Illumina technology based studies. We could use PippenPrep to do this, of which we have housed in the Suni Lab (#Suni!!!) Alternatively, we could run out the PCR on a gel extraction to cut them based on a ladder. Lastly, you could use metallic beads to isolate the DNA.
After size selection, DNA size samples would be normalized–bringing all of them to approximately the same concentration. The final step after that would be to combine all the size selected and normalized PCR products into one vessel. Then these samples wold be run on the in-house Illumina sequencer. Sequencing would take ~16 hours and if successful, would generate tens of millions of reads. These data would be run through a bioinformatics pipeline (looks at Dr. Zimmerman). Ultimately we would align these data with the published M. guttatus genome and call SNPs. We would use these SNPs to infer population differentiation using a metric like FST and assess population genetic diversity looking at things like the number of alleles, allelic diversity, etc.
Based on what we know about M. guttatus, geographically diverged samples will probably have larger population differentiation than those who are geographically closer to each other.
This lab we ran a test PCR to see if there was a successful library construction on our Mimulus guttatus samples using test PCR.
PCR I – Test Library Construction Success
NEB One-Taq 2x Master Mix – 80 uL
Forward Primer – 4 uL
Reverse Primer (PCR 026) – 4 uL
Pure H2O – 62UL
Total reaction volume 16 uL
PCR1 ws used on BIORAD #1/2 at 94 degrees Celsius for 2 minutes, then 20 cycles of 94 degrees celsius for 30 seconds, 60 C fr 30 seconds, 68 C for 45 sec, and then at 4-10 C at infinite hold.
We then ran the products of PCR I for each sample on a 1.5% agarose gel with a 100 bp ladder at 130 V for 40 minutes.
PCR II – to generate final Illumina sequencing library
10 PCR reactions were set up in 25 uL volume.
Phusion DNA polymerase 3.1 uL
5x Phusion HF buffer 62.5 uL
Forward primer (PCR1_X) 15.6 uL
Reverse primer (2-7) 15.6 uL
DNTPS 6.3 uL
DMSO 9.4 uL
Pure H2O 107.5 uL
Master Mix Total 220 uL
Total reaction volume 25 uL
These were pipetted with individual samples into PCR tubes and then vortexed. PCR2 was run on BIORAD #2 for a number of cycles. Then 2 uL of the products of PCR2 were run on a 1% agrose gel with a 100 bp ladder.
Results of PCR 1 and PCR 2 are below.
This lab, we performed a DDRadseq of Mimulus guttatus samples. DNA samples collected from various locations (see Table 1 for codes) were chosen and 6 uL of each sample was placed in a PCR tube and stored on ice. A master mix was created with 9.9 uL of CutSmart 10x bugger, 3.1 uL ECORI-HF enzyme, 1.32 uL of MPSI enzyme, and 18.7 uL of pure H2O. 3 uL was added into each sample’s DNA. Samples were sealed, vortexed, and then placed in an incubator at 37 degrees Celsius for 8 hours on a thermocycler with a heated lid set to 50 degrees Celsius.
Our previous samples, prepared in the same way above, were then used to perform the second step of DDRADseq. Samples were placed on ice and 1 uL of EcoRI’s were added (see Table 2) to each sample. A master mix was created with 4.4 uL of CutSmart buffer 10x, 14.3 uL of ATP 10 mM, 2.2 uL of T4 igase enzyme, 1.1 uL pure H2O, and 11 uL of working stock universal P2 MspI adapter. 3 uL of this master mix was added to the digested DNA. Samples were sealed, centrifuged, and incubated at 16 degrees Celsius for 6 hours on a thermocycler with a heated lid set to 50 degrees Celsius.
This lab, we added microsatellites to the previously prepared samples of M. guttatus in order to process the DNA and to understand genetic diversity within M. guttatus.
- 10x buffer
- F-Primer (MIRI70)
- R-Primer (MIRI70)
11 PCR reactions were prepared to accommodate 9 samples of M. gutattus. All components of the PCR reaction were kept on ice. 146.9 uL of ddH2O, 22 ul of 10x bufffer, 22 ul of MgCl2, 11 uL of BSA, 2.2 uL of dNTPs, F-primer, and R-primer, and 0.44 uL of Taq were added to make approximately 19 uL per reaction. 1 uL of each sample was added to an individual tube and vortexed with the reaction mix to be processed.
This lab, we simply ran our gels for the samples processed in Lab 07.
12 loading dye dots of about 1 uL were pipetted onto a sheet of parafilm. 3 uL of each sample was placed into its own dot. All dots were loaded into the gel alongside others’ samples and run for approximately 40 minutes.
This lab period utilized a modified version of Alexander et al. tube protocol for DNA extraction.
Materials and Methods
- 2.0 mL tubes
- Sterile 3.2 mm stainless steel beads
- Mimulus guttatus leaf tissue (preserved in silica)
- Tube rack
- Modified reciprocating saw rack
- p20 – p1000 pipets and pipet tips
- Grind buffer
- 3M pH 4.7 potassium acetate
- 1.5 mL tubes
- Binding buffer
- Epoch spin column tubes
- 70% EtOH
- Pure sterile water
I labelled 3 2.0 mL tubes with the codes RWCK 003 AJC, MAPL 005, and CATB – EVR. The first two samples were obtained by Alex, a graduate student working with Professor Paul. Three sterile 3.2 mm stainless steel beads were added to each tube and a small amount of leaf tissue from each sample was placed in their respective tubes. The tubes were placed in a rack, which then was placed in a modified reciprocating saw rack and was reciprocated on speed 3 for 40 seconds. The tubes were placed in the centrifuge for 18 seconds to pull plant dust down. 434 microliters of preheated grind buffer was added to each tube. The buffered grindate was incubated at 65 degrees Celsius for 13 minutes and 30 seconds and were mixed by inversion every three minutes. 130 microliters of 3M pH 4.7 potassium acetate was added and mixed by inversion. These were incubated on ice for 5 minutes and 5 seconds. Samples were then centrifuged at maximum force for 20 minutes. New 1.5 mL tubes were labelled with the aforementioned sample IDs and the supernatant was transferred to those tubes. Some precipitate was pipetted into sample 3. 1.5 volumes of binding buffer was added which was around 550 microliters for each of the samples. 650 microliters of the mixture was added to Epoch spin column tubes and centrifuged for 10 minutes at 15,000 rpm. The flow through was discarded in a hazardous waste container and then the process was repeated with another 650 microliters. The DNA bound to the silica membrane was washed by adding 500 microliters of 70% EtOH to the column and then centrifuged for 8 minutes. This step was repeated after discarding the flow through. The columns were then centrifuged for a final time for five minutes to remove residual ethanol. The collection tubes were discarded and the columns were placed in 1.5 mL microcentrifuge tubes that were labelled with the ID and date. 100 microliters of pure sterile H2O was added to each tube, which were allowed to stand for 5 minutes and then centrifuged for 2 minutes at 15,000rpm to elute DNA.
This lab period utilized Geneious, a program that performs various functions on DNA and protein sequence data, to analyze the data collected in Lab 02. Both the forward and reverse of EVR03 succeeded while EVR01, EVR02, and EVR04 did not return any usable data. To substitute, I am using the extra code BP03 which was reported as albacore tuna.
Forward and reverse primers of EVR03 and BP03 were obtained and put through Geneious and were assembled in their respective samples. A new folder was made and the documents were copied and de novo assembled. The sequences were trimmed at the ends where confidence was low and some corrections were made to ambiguous codes where there was a discrepancy between the base calls of forward and reverse primers. Consensus sequences were generated and the sequences were BLASTed and the top hits were compared against what the fish was advertised as. An alignment was built of the sequences with some of the hits–EVR03 was tested against 8 different hits while BP03 was tested against 9 different hits. The alignments were built with Multiple align and muscle alignment with default settings.
- For EVR03, it was listed as mackerel–the BLAST revealed that the sequence was in the Scombridae family and had a grade of 99.2% for both Thunneus obseus and Thunneus albacares, though I am inclined to believe it is T. obseus because it’s slightly higher than T. albacares in the BLAST details. For BP03, it was listed as albacore tuna. The BLAST revealed the sequence was closest to Thunnus alalunga, which is, in fact, the albacore tuna. This result had a grade of 99.7%.
- Polymorphisms in EVR03’s nucleotide alignments were found at sites 178 and 292. Ambiguous codes weren’t found in sites with polymorphisms. The first ten polymorphisms in BP03’s nucleotide alignments were found at sites 19, 31, 46, 277, 283, 301, 328, 352, 373, and 415. There were 18 polymorphic sites in all.
This class and lab period (12:45 to 17:00) was used as another field trip to collect samples of M. guttatus and observe them in their habitats. Two areas were visited: the Red Rock Beach Cold Spring and a lower trail path near Muir Woods.
The spring is located near Stinson Beach and potable water from the Mount Tamalpais watershed flows through three pipes located in the area. The constant flow of water means that even though the spring is located in a rather sunny and hot area (especially on the day of the lab where temperatures continued to climb well beyond a normal 70 degrees), M. guttatus is able to flourish there.
M. guttatus’s “landing strip” for bees is also pictured below.
Because it’s pollinated by bees and the area of M. guttatus is limited by its access to water, the populations of M. guttatus in the area are likely inbred due to that limitation.
The second area had much more access to water–even though the water level of the creek is lower due to the fact that we’re just getting into fall and the winter rains haven’t fallen yet, there’s still enough water to support various populations of M. guttatus and facilitating their gene flow through water and pollination.
Some populations were located in areas that could be flooded before they properly disperse their seeds, which introduces questions on effective population size vs. population size and how conservation efforts should approach species in different areas.
This lab is a continuation of Lab 02 and uses samples from that entry.
- PCR tubes
- Pipette tips
- Distilled water
- Sap 10x
- Loading dye
- Gel electrophoresis equipment
Part I – Gel Electrophoresis
PCR tubes containing the samples labelled with EVR01, EVR02, EVR03, and EVR04 were thawed. 16 loading dye dots of about 1 uL were pipetted onto a sheet of parafilm. 3 uL of each sample was placed into its own dot. All dots were loaded into the gel alongside others’ samples as shown in Table 1. The gel was run at 130 volts for 30 minutes, which can be seen in Figure 1.
Part II – PCR Cleanup
The number of PCR cleanups that would need to be done for the products in Table 1 was calculated. Reagents were placed on ice. 7.5 uL of EVR01, EVR02, EVR03, and EVR04 were pipetted into their own 0.2 uL PCR tubes which were labeled with their respective codes. The ExoSap master mix was made using the numbers calculated in Table 2. 12.5 uL of the ExoSap mix was added to each PCR product tube. The tubes were placed into a thermocycler and the EXOSAP program was started which ran for about 45 minutes. The PCR tubes were placed into a labeled tube rack and placed in the freezer.
Gel Electrophoresis Template: Genomic DNA PCR product
|Lane # (L -> R)
||Sample ID #
Recipe to clean-up one PCR reaction of 7.5 uL
Master Mix: Rxn: 1 Rxns: 13
H2O 10.59 uL 137.67 (138)
10x buffer (Sap 10x) 1.25 uL 16.25 (16.3)
SAP 0.44 uL 5.72 (5.7)
Exo 0.22 uL 2.86 (2.9)
Master Mix Total 12.5 uL 162.5
PCR 7.5 ul
Total Cleaned-up Volume 20.0 uL
This class and lab period (12:45 to 17:00) was used as a time for the class to go on a field trip to Mt. Tamalpais and observe the species Mimulus guttatus and its different forms it takes. M. guttatus, also known as the monkey flower, is a plant with a wide distribution range in the United States which has various forms adapted to different areas. This particular species has yellow flowers, which implies that its pollination syndrome is by bees.
Three areas were visited to observe M. guttatus forms. The first was Cataract Bridge, a shaded trail with a small creek running through it. Alex, one of Professor Paul’s graduate students, enlisted the class’s help to collect samples from this area, which were dried in a silica gel in order to preserve them better for processing. M. guttatus “likes to keep its feet wet” which means they’re generally found in areas with moisture, where they then put down runners. When sampling, young leaves were taken from plants that were at least a meter apart to ensure they weren’t the same plant. Brief discussion of how M. guttatus seeds were spread occurred–one way was likely through water flow. This is one of the methods Alex is using in his research: how molecular ecology can be used to analyze gene flow.
Slightly up from Cataract Bridge is a field that, in the heat, was largely dried up. Despite this, we observed an M. guttatus with its seed pods. Their seeds are quite small, but M. guttatus was able to survive with very limited water in this area.
Next, we observed M. guttatus in a serpentinite patch. Serpentinite occurs in areas where two plates meet (and it’s the state rock of California!) It occasionally allows water to seep up through when it is cracked, which is where some M. gutattus patches survived.
Lastly, we walked a path to find M. guttatus in a valley. Water flows down through the valley and enables green patches to crop up–we found M. guttatus among a patch of rushes and sedges which was surprisingly muddy despite the dry grasses and buckwheat surrounding it.