Below is the current best practices protocol for SWGA. We have historically run SWGA in 50 ul reactions.
Reagents
- 50 uM premixed primers (50 uM is the total primer concentration, not the concentration of each primer)
- 10x phi29 buffer
- 100x BSA
- 10 mM mixed dNTPs
- phi29 enzyme
- Molecular grade H2O
- Less than 50 ng total DNA
Reagent
|
Quantity
|
Final
Concentration
|
DNA
|
<50 ng
|
<1 ng/ula
|
Primer set (50 uM)
|
3.5 ul
|
3.5 uMb
|
Phi29 Buffer (10x)
|
5 ul
|
1x
|
BSA (100x)
|
0.5 ul
|
1x
|
dNTPs (mix, 10 mM each)
|
5 ul
|
1 mM
|
phi29 enzyme (10 U/ul)c
|
3 ul
|
0.6 U/ul
|
H2O
|
Fill to 50 ul
|
-
|
aSWGA relies on amplification for enrichment, and output DNA quantity does not increase linearly with input DNA quantity. For this reason we recommend using less than 50 ng of total DNA to start to ensure adequate amplification. The exception to this is when methylation dependent restriction enzymes are used.
bThis is the total primer concentration, not the concentration of each primer.
cphi29 enzyme should be added last to prevent any non-specific amplification.
bThis is the total primer concentration, not the concentration of each primer.
cphi29 enzyme should be added last to prevent any non-specific amplification.
Amplification Conditions
Ramp down from 35oCd to 30oC, 10 minutes per degree
16 hours at 30oC
10 minutes at 65oC
Hold at 4oC
dphi29 is not thermostable, it is therefore critical to ensure that the thermocycler starts at 35oC and not in hot-start mode.
dphi29 is not thermostable, it is therefore critical to ensure that the thermocycler starts at 35oC and not in hot-start mode.
Cleanup
Prior to cleanup add 50 ul of H2O to each reaction in the PCR tube and mix thoroughly. This decreases the viscosity of the final product and ensures that the DNA is properly mixed.
We purify the DNA using Agencourt AMPure XP beads, using a 1:1 volumetric ratio of beads to product.
