The reaction buffer mix was combined with template DNA and brought to a final concentration of 2x.
Thereafter, the wells were washed with PBS by cautious pipetting and the recovered buffer was combined with the GM previously recovered from the wells.
Thereafter, the lysed samples were removed from wells by vigorous pipetting, the wells were washed with PBS and the recovered buffer was combined with the lysate previously recovered from the wells.
Culture dishes were washed once with 200 μL of lysis buffer, which was combined with the lysate.
RNA in 100 mM citrate buffer pH 6.0 was combined with the ethanolic lipid solution through a T-mixer, and an additional buffer volume of 100 mM citrate buffer pH 6.0 was added simultaneously to the RNA/lipid suspension resulting in a 33% ethanol solution.
Briefly, 400 μL of blood lysate (200 μL whole blood + 200 μL RNA isolation buffer) was combined with an equal volume of acid phenol, vortexed, and centrifuged at 13200 g for 15 min. One half of the aqueous phase was transferred to a fresh tube and vortexed with 1.25 × volume of 100% ethanol.
To avoid starch interference and improve the RNA yield, 1 vol of Qiagen RLT extraction buffer was combined with an equal volume of the following solution: 2 % (w/v) CTAB (Cetyl Trimethyl Ammonium Bromide); 2% (w/v) PVP 40; EDTA 25 mM; Tris/HCl 100 mM (pH 8).
30 μL of a solution containing SDS loading buffer and mercaptoethanol were combined with 50 μg of sample protein and lysis buffer up to 100 μL.
The attached cells were gently rinsed twice with PBS, drained, and lysed at 4°C in 0.5 ml of lysis buffer with occasional vortexing for 30 min. The lysate was transferred to a clean tube (1.5 ml), and the remaining cell matter was washed off the surface of the flask with a further 0.5 ml of lysis buffer, and lysates were combined.
After a 2 min incubation in this buffer, the actin solution was combined with an equal volume of Arp2/3 complex and VCA materials in double strength KMEI buffer (such that the final solution was 10 mM imidazole pH 7.0, 50 mM KCl, 1 mM EGTA, 1 mM MgCl2, 0.5 mM DTT, with one half concentration of buffer G carrying over from the actin solution).
