BigDye Dilution Experiment
Three different reaction set ups were analyzed, 4 tubes for each condition:
#1: 0.5 ul BigDye V3.1; 0.2 ug pGEM (1 ul); 2 pmol M13F -20 (2 ul); 2.5 ul 5X buffer; 0.5 ul 5M Betaine; 5.5 ul water
#2: 0.25 ul BigDye V3.1; 0.2 ug pGEM (1 ul); 2.5 pmol M13F -20 (2.5 ul); 3.75 ul 5X buffer; 0.5 ul DMSO, 4 ul water
#3 2 ul BigDye V3.1; 0.2 ug pGEM (1 ul); 5 pmol M13F -20 (5 ul); 4 ul 5X buffer; 1 ul DMSO, 7 ul water ( our standard reaction set up contains 2 ul BigDye and 3 ul 5X buffer, BigDye itself is suspended in 2.5X buffer)
Conditions #1 and #2 are 12 ul reactions, #3 is a 20 ul reaction
Samples were cycled at 98° hot start for 1', then underwent 30 cycles of 98° 10"--50° 10"--60° 4', then finished with 72° for 1 minute and 4° forever.
Samples were cleaned with magnetic beads; #1 and #2 samples were brought to 20 ul volume before adding the magnetic beads, then ultimately resuspended in 20 ul (it takes some flicking to get the water to resuspend all the beads), #3 was resuspended in 50 ul. 10 ul of each was then mixed with 10 ul water in the sample plate, then put on the machine (SOP). For each set (4 diff tubes for each) the length of read (LOR) with Q values >20 and the avg. signal strength for each base is presented:
LOR G A T C
#1 993.5 ± 13.1 4087 ± 507 3281 ± 414 3083 ± 371 4865 ± 616
#2 950.8 ± 35.0 683 ± 264 607 ± 196 657 ± 206 892 ± 294
#3 961.0 ± 24.5 2070 ± 654 1927 ± 592 1503 ± 487 2232 ± 726
Basically this shows that 0.5 ul (#1) performs about as well as 2 ul #3: the lower signal strength with 2 ul is roughly proportional to the greater volume used to resuspend the beads after cleanup. Even 0.25 ul can give decent data, although there is a substantial fall off in signal strength.