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Lab 11: Double Digest and Ligation

November 12, 2019


Double Digest 

In our lab, we set out to digest our Mimulus guttatus samples by beginning our double-digest restriction associated DNA sequencing. I labeled 8 PCR tubes (1-8) that contained 6μL of sample each and stored the tubes on ice. We then prepared a master mix that contained; 9.90μL of CutSmart buffer 10X, 3.1μL of EcoRI-HF enzyme, 1.3μL of MSPI enzyme and 18.7μL of pure H2O for a total of roughly 33.0μL. The master mix was capped in the tube, mixed well, centrifuged, and stored on ice. I then added 3μL of master mix to all 8 PCR tubes with the DNA samples. The tubes were sealed, vortexed, placed in the centrifuge and incubated at 37° C for 8 hours on a thermocycler with a heated lid set to 50°C.


Ligation

Next, we were given the task of completing an adapter ligation for RADseq. Before making the Master Mix, I added 1μL of the working stock EcoRI adapter, EcoRI 8 and EcoRI 9, directly to my digested DNA samples which were sample ID# 23 and 24. I was then able to create the adapter ligation master mix for RADseq. The master mix contained; 4.4μL of CutSmart buffer 10X, 14.3μL of ATP, 2.2μL of T4 Ligase, 1.1μL of pure H2O and 11.0μL of Universal P2 MSPI adapter for a total volume of 33.0μL. After it was complete, I added 3μL of the master mix to the digested DNA. The tubes were sealed, vortexed, placed in the centrifuge, and incubated at 16°C for 6 hours on a thermocycler with a heated lid set to 50°C.

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Lab 10: PCR Reactions

October 5, 2019


We started our lab session by first collecting our tubes of Mimulus guttatus DNA. We obtained 12 1μL tubes and labeled them ‘QS 1-12.’ QS stands for our group name ‘Queen Salmon.’ We were sure to write down the corresponding specimen ID number to the tube number assigned to each group member.

We then proceeded to make the master mix that was a key part of this PCR reaction. We were given the amount of each ingredient that was made for one reaction. Since we had 12 PCR reactions to complete, we decided to multiply these values by 18 to account for each reaction as well as any error that could occur. The ingredients consisted of; 240.48μL ddH2O, 36μL 10x buffer, 36μL MgCl2, 18μL BSA, 3.6μL dNTPs, 3.6μL F-primer, 3.6μL R-primer, 0.8μL Taq. This mixture was then vortexed to make sure that the ingredients were properly mixed together.

We then took the new labeled tubes and added 19μL of the master mix as well as the 1μL template from our individual samples. The tubes were then vortexed for a few seconds before being stored on ice.

 

 

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Lab 9: Gel Electrophoresis

October 29, 2019


We began our electrophoresis lab by gathering our Mimulus guttatus DNA samples from our last lab. I first started by dotting out 16 loading dye dots on a sheet of parafilm. I loaded 1 µl dye dots using a 20 µl micropipette. We then pipetted 3 µl of each PCR product into its own dot. After all the product was pipetted into the loading dyes, we then set the pipette to 5 µl and loaded the dots into each well. We then ran the gel at 130 volts for 30 minutes.