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 |
- Add all reagents to master mix except for SYBR Green and enzyme
- Heat up bacteria at 90 C for 2 min to lyse.
- While heating up, add SYBR Green to master mix
- Cool bacteria at 40 C for 2 min and then put on ice
- While cooling at 40 C, add enzyme to master mix and mix thouroughly
- Add 10 uL of bacteria in agarose to 4 tubes, 10 uL of bacteria in H2O to 2 tubes (+ control) and 10 uL .75% agarose to 2 tubes (NTC). Put tubes on ice.
- Add 10 uL of master mix to each respective tube. Final conc. of agarose is .375%
- Perform real time MDA with the opticon machine
Results
The following image shows an inconclusive result. While 1.5 uL of beads did work the best, it is unclear why .5 and 1 uL did not work. The expt will have to be repeated, possibly on a smaller scale to ensure proper mixing of reagents. Another note is to make sure the agarose stays in its liquid phase.