ISSR Analysis and Concatenation of Alignments from EPIC Markers

November 14, 2018

The wet lab consisted of two parts: the ISSR Analysis of the Lupinus aboreus , and the concatenation of Mimulus cardinalis alignments from the four EPIC markers.

Part one of the lab, the ISSR analysis of the Lupinus aboreus samples, consisted of running the PCR reactions generated from previous labs. First, approximately 1­-µL of loading dye was added to each of the PCR tubes, making sure to switch pipette tips between tubes. After completion, 5-µL of each sample was loaded onto the large 2% agarose gel by a tablemate. 48 samples from our table was loaded onto one of the rows of 50 wells, with the remaining 12 samples occupying the wells in the last row, following the group before us. The first and last wells of each row were meant to be saved for the ladder. Having established the order of our samples, it was determined that my samples would be loaded last. Thus, 12 of my samples should be on the last row with 3 on the row with my tablemates. The gels were then run on a low voltage of 20 volts. Due to circumstances, we did not get to score the banding patterns of the gel, though the protocol for identifying the bands would be to mark a ‘1’ if a band was present, and a ‘0’ if a band was absent.

The second part of the lab was performed on Geneious. Having already identified heterozygous sites and built alignments from previous class data, several additional sequences were downloaded and imported into Geneious under the folder ‘5525 Consensus Sequence ver2’. These additional sequences were found on Canvas as the file ‘EPIC_5525_New’. Having imported the new marker documents, each forward and reverse pair was assembled together using the function ‘De Novo Assembly’. These consensus files were edited via trimming of bad starts and ends and relabeling of heterozygous or poorly called sites. Consensus sequences were generated from the edited forms of these assemblies. Using the ‘Batch Rename’ function under ‘Edit’ and selecting all the newly formed consensus sequences, 33 characters were erased to rename the files. The files were then copied and pasted into the ‘5525 Consensus Sequence ver2’ folder containing the previous alignment. All the consensus sequences were then selected (consisting of old and new) and a new Muscle nucleotide alignment was generated using default settings. Additional edits such as trimmings of bad ends and starts were performed before the file was saved and closed. The new alignment was then renamed to be ‘5525_Nucleotide alignment_YUCHUH’ containing a total of 33 sequences. A new folder was created under ‘Local’ called ‘Concatenate’. The new alignment was copied and pasted into this new folder. It was also exported as a geneious file and uploaded to Canvas under the assignment ‘EPIC ALIGNMENTS’. From the variety of marker alignments under the module ‘EPIC ALIGNMENTS’, three marker alignments generated by peers were imported into the ‘Concatenate’ folder. All four files in the ‘Concatenate’ folder was selected and the function ‘Concatenate Sequences or Alignments’ under ‘Tools’ was used. A file containing all four files was created called ‘Concatenated 1’. This file was then used for exercises later detailed.

Plant Population Genetics III

November 7, 2018

 

Preparation of Lupinus arboreus samples for PCR amplification using interspersed simple sequence repeat (ISSR) primers was performed during this phase of the project. Using ISSRs, the ultimate objective is to determine whether populations can be grouped more closely through geographical proximity or flower color. The following procedure was used to prepare 1:10 dilutions of the five Lupinus arboreus samples provided:

  1. Five samples of Lupinus arboreus samples were given (PRA01 to PRA05).
  2. Fifteen 1.5-mL tubes were obtained and labeled with the ID code, three new tubes for each original sample.
  3. 45-μL of H2O was placed into each newly labeled tube.
  4. 5-μL of each sample was pipetted into three of the correspondingly labeled dilution tubes, switch pipette tips across samples.
  5. One of each dilution tube was placed into a separate rack for dispersal.

After completing the dilution process, the preparation of the template DNA and Master Mix (MM) was performed:

  1. Two 0.2-mL 8-tube PCR strips were obtained for the fifteen reactions.
  2. Completed the PCR sheet by indicating the tube number and corresponding sample ID.
  3. Tubes were labeled on the cap with information matching that of the PCR sheet, including additional details such as primer number and initials written along the sides of the tubes
  4. 1-μL of diluted DNA from each sample was pipetted into the corresponding PCR tubes, following the order of the PCR sheet. Pipette tips were changed between samples.
  5. Once completed, the lid was gently closed atop the tubes and the strips were placed on ice.
  6. To create the master mix, reagent amounts were calculated for 70 reactions, resulting in a MM of 875μL of ddH2O, 210μL of 10x buffer + Mg, 70μL of BSA, 140μL of dNTPs, 17.5μL of primer OMAR, and 17.5μL of Taq (provided by the Professor). Reagents were added to a 1.5-mL tube labeled MM for Master Mix in the listed order.
  7. 19-μL of the MM were pipetted into each PCR tube, pipetting up and down to mix the MM with the diluted sample. Tips were changed for each sample, and the last tube (tube 16) contained only the MM to act as a negative control.
  8. PCR tube strips were capped tightly then placed on a rack for PCR.

Plant Population Genetics II – Analysis of EPIC Markers

October 31, 2018

Due to unfortunate circumstances, there were no forward or reverse reads for the four EPIC markers used in the class data that included the twenty-five samples of Mimulus cardinalis. Thus, data from previous classes was used to score for polymorphisms, code for heterozygotes, and assemble EPIC marker alignments. The following protocol was performed:

  1. A new folder named ‘Plant Population EPIC Marker 5525’ was created under the ‘Local’ category.
  2. 5525_Forward_Reverse file was downloaded from Canvas and imported into Geneious inside of the ‘Plant Population EPIC Marker 5525’ folder, creating a new folder named EPIC_5525_Forward_Reverse.
  3. A muscle alignment was generated by selecting all the sequences and selecting ‘Multiple Align’.
  4. Ends were trimmed on the starts and ends of all sequences from where the good reads began.
  5. Polymorphisms were scored and the columns recorded.
  6. Heterozygous sites were identified by comparing whether there were dual clear peaks within both forward and reverse reads for the same sample. These sites wee recorded.
  7. Heterozygous sites were re-coded with the symbols according to the IUPAC Ambiguity Code list by selecting the heterozygous site and replacing the nucleotide with the corresponding letter.
  8. After all heterozygous sites were re-coded, the document was saved, making sure to apply changes to the original sequences.
  9. An assembly sequence was generated for each forward and reverse reads pair by selecting the pair and using ‘De Novo Assembly’ with default settings.
  10. Open each new assembly document and trim sequences at the start and ends where the good reads begin. Also edit any areas where nucleotides may be ambiguously marked with an N on one read, but clear on the other read. Replace the ambiguous nucleotide with the clear nucleotide.
  11. Double check heterozygous sites and ambiguities, then save the document. Repeat for all assembly sequences of each sample.
  12. All assembly documents were selected and ‘Generate consensus sequence’ was clicked then a sequence list was created, generating a new file named ‘Consensus sequences’.
  13. ‘Consensus sequences’ was selected and a new folder named ‘5525 Consensus Sequences’ was created by click ‘Sequence’ then ‘Extract sequences from list’, followed by ‘Extract to a new subfolder’.
  14. All files were selected in the new folder the batch was renamed by removing 33 characters from the end.
  15. The outgroup (TG0248…) was copied from the ‘EPIC_5525_Forward_Reverse’ folder and pasted into the ‘5525 Consensus Sequences’ folder. The name was then edited to ‘TG0248’.
  16. All sequences were selected and used to build an alignment using Multiple Align, Muscle, and default settings.
  17. The alignment was opened and double-checked for possible additional edits then saved and renamed to ‘5525 Nucleotide alignment’.
  18. A MrBayes tree inference was run on ‘5525 Nucleotide alignment’ using the following parameters: HKY85 substitution model, gamma rate variation, TG0248 outgroup, 3,000,000 chain length, 500 subsampling frequency, 300,000 burn-in length, and 24,278 random seed. The results are shown below: 

Data/Results 

Based on the first nucleotide alignment created with all forward and reverse reads present, there a number of polymorphic sites as well as heterozygous sites. Some even overlapped and were both polymorphic and heterozygous sites. There were twelve apparent polymorphic sites, exhibited in columns 41, 48, 56, 74, 86, 140, 191, 239, 303, 315, 329, and 334. For the heterozygous sites, five were identified, shown in columns 86 (C/T), 140 (C/G), 191 (A/G), 239 (C/G), and 334 (A/C). However, in both cases, there may have been more that were not identified due to carelessness or human error. Four sites exhibited both polymorphism and heterozygosity. These columns included 86, 140, 191, and 334.

According to the inferred tree, it appears that the clade at the top comprised of samples JP1132, JP1158, JP1133, JP1134, JP1156, JP1228 was strongly supported (100%), as well as the clade that grouped JP1152 separately from the rest of the 19 samples (approximately 99%). It is possible that the clade at the top represented populations that were geographically closer, due to the degree of their genetic similarity and the posterior probability value. However, since the bottom clade only portrayed a posterior probability of approximately 51%, there may be other factors aside from geographic proximity that contribute to the lower support value obtained.

Plant Population Genetics – DNA Amplification

October 24, 2018

Following the previous lab session, an agarose gel electrophoresis was run using the extracted DNA to confirm the presence and quality of template DNA from the samples. Using exon-primed, intron-crossing (EPIC) markers for the primers, the obtained template DNA was amplified via PCR. Two additional tubes of sample DNA were made for each table such that each table had a copy of each sample.

Specifically, the following procedure was performed for the gel electrophoresis:

  1. Loading dye was pipetted onto a piece of Parafilm in 3-μL increments, following the placement of the wells on the agarose gel.
  2. 1-μL of template DNA for each sample was pipetted into a 3-μL spot of loading dye according to the order portrayed below. 
  3. The total 4-μL volume of dye and template DNA was then pipetted into three separate wells on the gel.
  4. The gel box was then assembled with the correct alignment of positive and negative electrodes, then run for approximately 25 minutes at 140 volts.
  5. As the gel electrophoresis was running, two separate tubes were labeled for each sample, making a total of six newly labeled tubes portraying the sample IDs.
  6. 20-μL of DNA from the original tube was placed into each of the two tubes corresponding to the same sample ID. Repeated this step with all three samples, ensuring that pipette tips were changed when moving on to a different sample.
  7. Gel apparatus was disassembled, and the gel was removed from the apparatus onto a UV imagining machine.
  8. Picture of DNA bands was obtained.

After completing the gel electrophoresis, preparation of the template DNA and creation of the master mix for the PCR amplification was completed according to the steps listed below:

  1. Eight Mimulus cardinalis template DNA samples were obtained from the class pool.
  2. Tube numbers and sample IDs were recorded according to the table shown.
  3. One strip containing eight 0.2-mL PCR tubes were obtained and labeled with the tube number, specimen ID, according to the table, in addition to the primer and initials.
  4. 1-μL of template DNA was pipetted into each PCR tube, ensuring that the sample matched the tube number and changing tips in between samples.
  5. After transfer was completed, the PCR tubes were closed and placed on ice.
  6. A 1.5-mL tube was labeled as MM (MasterMix), and the reagents depicted in the image below were added in the following order: distilled H2O, 10x PCR buffer (including MgCl2), BSA, dNTPs, Forward primer, and Reverse primer. 
  7. This solution was then placed on ice until Professor Paul pipetted the required amount of Taq polymerase into the MM tube.
  8. 19-μL of the MM was then added to the PCR tubes.
  9. PCR tubes were closed and given to Professor Paul for PCR amplification.

 

Plant Population Genetics – DNA Extraction

October 17, 2018

 

Using molecular markers to obtain a greater understanding of the degree of population differentiation and gene flow between populations of mimulus cardinalis, this phase of the experiment focused on the extraction of DNA from plant tissue. Mimulus cardinalis from various areas were collected and preserved in test tubes using silica, which quickly dried the plant tissue to enable better DNA preservation. Three samples of Mimulus cardinalis were provided in addition to their sample identification codes: JP1292, JP1304, and JP1302. DNA was extracted from these samples according to the following procedure:

  1. The species name, sample IDs, and location (when available) was recorded on a chart, such as the one shown below.
    Sample ID Location Species
    JP1292 Mimulus cardinalis
    JP1304 Mimulus cardinalis
    JP1302 Mimulus cardinalis
  2. The IDs were used to label three 2.0-mL tubes with a sharpie pen on the tube’s side and cap.
  3. Three sterile, stainless, 3.2-mm metallic beads were added to each tube.
  4. Taking the tubes containing the samples, a small portion of dried leaf tissue was broken off (approximately a fingertip’s amount) and placed into the 2.0-mL labeled tube. To avoid contamination, the tweezers were cleaned and wiped after transference of each sample. Repeat with all three samples.
  5. The tubes were lined on a tube rack along the middle section and mounted onto a modified reciprocating saw rack.
  6. Ensuring that the blade was secured and locked into the saw, the plug was inserted into the outlet and the saw was powered on and left for 40 seconds on speed 3.
  7. The tubes were centrifuged for 15-20 seconds — making sure the centrifuge was balanced — to bring the powdery plant dust down from the tube caps.
  8. 434-µL of preheated grind buffer was pipetted into each tube, switching filtered tips after each sample to avoid possible contamination events.
  9. The tubes were fixed onto floaters inside a 65°C water bath for ten minutes, where they were inverted every three minutes to mix tube contents.
  10. 130-µL of 3M, pH 4.7 potassium acetate was added into each tube, switching filtered tips after each sample. Tubes were inverted several times to induce mixing then placed on ice for five minutes.
  11. Afterwards, centrifuge tubes for 20 minutes.
  12. New 1.5-mL tubes were labeled with IDs.
  13. Tubes were retrieved from the centrifuge, displaying stark contrast between supernatant and pellet/precipitate. The supernatant was transferred into the newly labeled 1.5-mL tubes corresponding to each sample, taking care to avoid pipetting precipitate.
  14. After recording the approximate amount of supernatant obtained and comparing with the desired end volume of solution, 600-µL of binding buffer was added to each sample to reach an approximate final volume of 1.5-mL in each sample. Filtered tips were changed after transference of each sample.
  15. 650-µL of the solution within each 1.5-mL tubes was pipetted into the Epoch spin column tubes, switching tips in between, then centrifuged for ten minutes.
  16. The liquid that flowed through was discarded, and the remaining volume of solution within the 1.5-mL tubes were pipetted into the corresponding column tubes and centrifuged, making sure to discard the liquid flow-through once again.
  17. 500-µL of 70% EtOH was added to each column tube and centrifuged for eight minutes to allow the liquid to pass through and wash the DNA bound to the silica membrane. The liquid that passed through was discarded. Repeated this washing step with EtOH twice.
  18. Placed tubes in centrifuge for another five minutes to remove residual EtOH.
  19. Labeled new 1.5-mL microcentrifuge tubes with IDs and date, then placed the columns (blue tubes, top part) into these sterile tubes while discarding the collection tubes (transparent tubes, bottom part).
  20. 100-µL of preheated pure H2O was added to each tube through columns, left alone for five minutes, then centrifuged for two minutes to elute the DNA. Columns were then discarded and the microcentrifuge tubes holding extracted plant DNA were relinquished to the instructor.

Some difficulties occurred with the operation of the centrifuge, though this occurrence did not affect the extraction steps, and only slightly prolonged the length of this experimental phase. The extracted DNA solutions appeared to be of varying shades of brown-green despite belonging to the same species, which may possibly be attributed to the difference in the populations’ environments and habitats.

Geneious II

October 11, 2018

During this lab session, Geneious was used to infer phylogeny trees for the 25 species of Actinopterygii that were chosen from the NCBI nucleotide database in comparison to the three sequenced nucleotide samples from the previous labs. Putting these together in one folder, a multiple alignment of all 28 sequences was created. Then, for better comparison between sequences, nucleotides tides were deleted from the beginnings and ends of the sequences to ensure equivalency in segment lengths using the shortest sequence as a basis for deletion. Six sequences did not fully match the homologous nucleotide sites of the others, so it was presumed that the misalignment may have been due to the sequences being the reverse complements. However, even after reversing the sequences and once again comparing all of the strands together, this misalignment was not solved. So, the sequences that did not align were removed from the overall alignment (though a different six sequences that matched the others were added later for the long-run analysis), which resulted in a end total of 22 sequences within that multiple alignment.

The next step was to install the jModelTest2 program in order to choose the best model of molecular evolution. Once the program was installed, the multiple alignment was exported in a Relaxed Phylip format and opened in the jModelTest program. The jModelTest then ran AIC and BIC analyses to generate likelihood scores for 88 models of molecular evolution to find the best model for the particular data set. Results from AIC analysis during the initial run of the experiment portrayed the best model to be TPM2UF + I + G, which did not correspond to the best model determined by BIC. BIC analysis showed HKY + G to be the best model of molecular evolution.

Afterwards, several hypothetical phylogeny trees were constructed based on results from Bayesian inference, maximum likelihood, and PHYML. Using the parameters outlined by the jModelTest2 and Bayesian inference, a short-run analysis was performed. The resulting posterior output graph did not have much of a shape or bars in general.

A second, longer run of the Bayesian inference using the same data was performed. The graphs obtained for this longer-run analysis showed a better distribution in terms of the bar graph shape and number of values. The predicted tree outcomes were very similar in terms of clades, but varied slightly in regards to the length of branches for specific sister taxas.

A maximum likelihood inference of a phylogenetic tree was also generated using the RAxML plugin on Geneious. Following the parameters of the evolutionary models determined by jModelTest 2 and choosing ‘Rapid bootstrap with rapid hill climbing’ , 100 trees were generated within one document. A consensus tree was then built out of these bootstrapping trees. However, the tree obtained was significantly different compared to the first analysis run with Bayesian inference since not only were many of the clades different, but the predicted max likelihood tree showed a significant amount of polytomy.

Finally, the last method tested used PHYML, but with the HKY85 model of molecular evolution  and the final Bayesian tree. However, despite running PHYML multiple times, the plugin seemed to have difficulty running and stays at 0% for over 15 minutes. Therefore, I was unable to obtain conclusive data using this particular method of computing maximum likelihood.

Geneious I

October 3, 2018

The introduction to Geneious lab segment illustrated the methods of utilizing the Geneious program. After completing the installation process, several sequences from the fish samples amplified in the previous labs were tested to identify whether the labels from the restaurant matched the information presented by the genomic data. These were the steps followed to complete the exercises using Geneious:

  1. Retrieved forward and reverse reads from Canvas and installed the files into Geneious.
  2. Copied and pasted a corresponding reverse read into the same folder as the forward read (eg. YH01_fwd, and YH01_rvs in same place).
  3. Assembled both forward and reverse reads onto same document using “De novo assemble”. Document was then named “YH#_ASSEMBLY”.
  4. Deleted any unreadable end bases.
  5. Modified bases that were illegible by comparing forward and reverse reads (eg. deleted unreadable bases on reverse strand and replaced it with legible base shown at homologous nucleotide site on forward read).
  6. Saved modified version of consensus sequence.
  7. Generated consensus sequence, which created new document under name “YH#_ASSEMBLY consensus”.
  8. Used BLAST on consensus sequence to search NCBI database for similar sequences.
  9. New folder of sequence matches appeared on menu.
  10. Identified whether sequenced sample matched labels provided at restaurant.

To build an alignment, these steps were taken:

  1. A new folder, named Fish Barcode Test Alignment, was created to host the barcode alignment documents.
  2. YH01_ASSEMBLY consensus sequence was copied and pasted into the newly created folder.
  3. 5 BLAST results within the top 100 hits for YH01 consensus sequence were selected and copied.
  4. The selected matching sequences were pasted into the Fish Barcode Test Alignment folder.
  5. All documents inside of the new folder were selected and the ‘multiple align’ function was used.
  6. A new document consisting of the nucleotide alignment of the five selected results and YH01 consensus sequence was generated.
  7. Polymorphisms were identified.

Due to unfortunate circumstances, the only samples sequenced using Geneious were YH01, YH02, and ARA01 (named YH_ARA01). YH01 was the sample of Tuna as depicted by the figure above, whereas YH02 consisted of a sample of Escolar. YH_ARA01 was an assigned sample of Yellowfin Tuna, as a replacement for the other samples that were unable to be properly amplified. YH01 and YH_ARA01 matched the species being served, though YH01 was not labeled as specifically. However, both samples were sequence-referenced through BLAST and the highest match percentages indicated that the species was Thunnus albacares, which was Yellowfin Tuna. On the other hand, YH02, the sample of Escolar, revealed a mixture of Thunnus albacares, and two different bacteria species. YH02 was sequenced three times, and broken into consensus sequences of YH02A, YH02B, and YH02C. BLAST results for YH02A matched the sample with Thunnus albacares, despite the differences in the sample’s appearance (Escolar versus Tuna), which indicated a degree of contamination between samples YHo1 and YH02. In addition, sequence YH02B matched most closely – though the percentage was fairly low – to Pesudomonas ludensis, a bacterial species that causes spoiling of milk, cheese, meat, and fish. Similarly, YH02C yielded a high match percentage with Pesudomonas fragi, a species of bacteria generally responsible for daily spoilages. YH02B and YH02C showed that not only was there contamination between samples, but also preservation issues regarding the sample of Escolar obtained.

According to the alignment built with YH01_ASSEMBLY consensus sequence, there were approximately 16 polymorphic sites (nt 681, 666, 426, 417, 375, 303, 288, 285, 279, 33, 25, 21, 12, 9, 6, 3) among the five chosen BLAST results. The first ten polymorphic sites were at positions: 3, 6, 9, 12, 21, 25, 33, 279, 285, and 288.

 

Field Trip II to Mt. Tamalpais

The second field trip consisted of an adventure through the winding paths of Mount Tamalpais. After passing through the Alpine Dam following the hiking trail, we climbed down a steep hill to find an area where the red-flowering mimulus cardinalis could have inhabited. The mimulus cardinalis is a flowering plant that generally uses hummingbirds as pollinators to reproduce.

 

 

 

 

 

The habitat consisted of a small, rocky, valley-like clearing with a small stream running through the side. Although the environment seemed to be a difficult place to inhabit, if a population of mimulus cardinalis was able to properly establish itself within this area, there would be significantly less competition due to the harsher conditions like the rocky bedding and limited sunlight. However, since the mimulus cardinalis’s main pollinators are hummingbirds, this area is still fairly accessible and may encourage reproduction despite the environmental conditions. Unfortunately, there were no populations that were spotted around this area, though there was interesting display of what appeared to be a ladybug festival.

We then moved across the bridge towards the other side of Mt. Tamalpais to another destination that usually had a population of mimulus cardinalis. The habitat was a small indentation at the side of the mountain, which was very accessible by pollinators, and surrounded by damp soil with a fair amount of sunlight. However, since the area was so small, there is a high possibility of in-breeding within this population of mimulus cardinalis compared to populations that inhabit larger environments.  

Afterwards, in order to observe the population of lupinus aboreus with purple flowers, we ventured to the other side of the mountain facing Stinson Beach. Atop the cliff-side, we were able to spot many individuals of purple-flowering lupine. The environment these individuals faced was very different compared to the populations at Pescadero Beach. While the yellow-flowering lupinus aboreus at Pescadero Beach had sandy soil, lots of sunlight, and fairly strong winds, the lupine at Mt. Tamalpais seemed to have little to no sunlight due to the heavy fog covering that side of the mountain, dry, grainy soils, and cold winds. The individuals at Mt. Tamalpais resembled the size of lupine at the cliff of Pescadero Beach, and appeared slightly smaller than those within the marshy area. The flowers also seemed to grow close to the ground in comparison to those at Pescadero.

                                      

Field Trip I to Pescadero State Beach

 

The first field trip on September 19th took the class to Pescadero State Beach, a coastal area approximately an hour away from San Francisco. We observed populations of Lupinus aboreus, a species of lupin that grows mainly along the coast of California. The Lupinus aboreus plant resembles a bush, growing relatively close to the ground with green finger-like leaves. However, the flower color of the Lupinus aboreus varies depending on its location, though there were only yellow flowers seen during the trip.

 

 

 

 

 

 

 

The coastal population that grew right next to the ocean atop a small cliff did not display any flowering, and the size of each plant seemed to be slightly smaller than the population that was growing further inland several meters. These small variations may be due to the fairly harsher conditions that these individuals faced, such as fierce winds and sandy soils compared to the more “sheltered” environment for those residing inland.

Finger-like leaves of Lupinus aboreus

Lupinus aboreus in the marshy inland

 

 

 

 

 

 

 

After crossing through the beach and under the bridge, we reached the population that was growing in the marshy inland near the beach. The individuals of Lupinus aboreus in this area displayed not only yellow flowers, but also seed pods that resembled edamame. The conditions seemed less harsh than the population growing by the beach as there was little wind, plenty of sun, and fairly moist soil.

 

 

The Sushi Test (cont.)

Subsequent to amplifying the genomic DNA (gDNA) collected from the fish samples, another gel electrophoresis was run, following the same steps as before, to see if specific bands were present. Unfortunately, some of the samples did not present the proper bands necessary to move onto the next procedure for PCR clean-up.

Another attempt was made by following the previous week’s steps for preparing the master mix and amplifying DNA via PCR. On the other hand, this would have been the protocol performed after obtaining the desired PCR results to clean up the amplified DNA and send for commercial sequencing:

  1. Prepare an ExoSap master mix containing water (10.59 μl), Sap 10x (1.25 μl), SAP (0.44 μl), Exo (0.22 μl), making sure that the reagents remain iced during preparation. The portions for each component should correspond with the numbers of reactions to be performed plus an additional amount covering for pipetting mistakes.
  2. Obtain a bucket of ice.
  3. Indicate and label the new PCR tubes to be used, then pipette 7.5 μl of each PCR product into the tubes.
  4. Pipette 12.5 μl of the ExoSap master mix into the newly labeled PCR tubes.
  5. Put the tubes in a thermocycler and start the ExoSap program, which should complete after approximately 45 minutes.
  6. Place the PCR tubes into a tube rack and put them in a freezer.

 

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