Jeff:LabNotes/Microbiome/2009-9-21
Jump to navigation
Jump to search
MDA in an Agarose base
See what the optimum volume of beads is for MDA.
Procedure
- Filter LMT agarose using 5 um pores
- Perform a serial dilution of bacteria in .75% LMT agarose to 1250 cell/uL. Also dilute in water for + control.
- Scale up the volume for MDA so that it 3x a normal reaction so the agarose is easier to work with
- Since 10 uL of template will be used, no water will be used in the master mix to keep the concentrations constant
- Want to vary the volumes of beads to see which works best
- The following volumes were used
Reagent | ' | ' | ' | ' | ' | ' | ' |
H20 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
.75% LMT Agarose | 12.6 | 12.1 | 11.6 | 11.1 | 10.6 | 10.1 | 9.6 |
C12 bound to bead | 0 | 0.5 | 1 | 1.5 | 2 | 2.5 | 3 |
N6 free primer | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
Buffer | 6 | 6 | 6 | 6 | 6 | 6 | 6 |
NTPs | 2.4 | 2.4 | 2.4 | 2.4 | 2.4 | 2.4 | 2.4 |
SYBR Green 2X | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
Phi 29 Enzyme | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
Total | 30 | 30 | 30 | 30 | 30 | 30 | 30 |