Microbio spin p 30 tris chromatography columns

A volume of 2.5 μl of gDNA from the blastocyst digestion as described above was used for whole genome amplification (Lucigen, Sygnis TruePrime WGA Kit #SYG380100 or Expedeon, Trueprime WGA Kit #380100). Kit instructions were as follows: 2.5 μl of buffer D was added to each gDNA sample and allowed to incubate for 3 min to denature the gDNA. The 2.5 μl of buffer N was then added to neutralize the denaturation reaction. Amplification mix was then added as follows: 26.8 μl of dH₂O, 5 μl reaction buffer, 5 μl dNTPs, 5 μl enzyme 1, and 0.7 μl enzyme 2. Samples were then incubated at 30°C for 6 h then heat inactivated at 65°C for 10 min. Samples were purified using MicroBio‐Spin P‐30 Tris Chromatography columns (BioRad, Hercules, CA). Columns were inverted to resuspend the gel matrix, then centrifuged at 1000 × g for 2 min to remove the matrix. Columns were placed in 1.5 ml microcentrifuge tubes and 20 μl of WGA sample was applied to the center of the column. Sample was centrifuged for 4 min at 1000 × g. Samples were then placed in −20°C freezer until further use. To check the fidelity of the WGA, a standard curve was constructed from known amounts of gDNA (Figure S3) and the template appears to amplify with high fidelity (R² = 0.9996).

Dulbecco's modified Eagle's medium (DMEM), Opti-MEM, 0.25% trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA). Fetal Clone III (FC) was from HyClone (Logan, UT). Complete-mini EDTA-free protease inhibitor cocktail tablets were purchased from Roche. Micro Bio-Spin P-30 Tris chromatography columns were purchased from Bio-Rad. Dithiothreitol (DTT), Dithiobis succinimidylpropionate (DSP), N-ethylmaleimide (NEM) and anti-FLAG M2 agarose beads were purchased from Sigma (St Louis, MO).