2/16/09
MDA testing with diluted human gDNA[edit]
Reagents[edit]
1. Nuclease free H20
2. 2M Tris buffer
3. 1N HCl (diluted from 12N HCl- 1 mL 12N HCl + ll mL H20)
4. 5M KOH (prepare fresh in 1.5 mL tube)
5. 2X SYBR Green (diluted from 10000X)
6. 200 uM primer
7. Diluted DNA
Materials[edit]
1. 2 strips of PCR tubes with caps
2. .6 mL tubes for master mix
3. 1.5 mL tubes
4. cool rack
Preparation[edit]
Thaw out all the reagents in Phi29 enzyme kit besides the actual enzyme.
gDNA Template Dilution
The following dilutions of gDNA were made
1. 1 ng/uL: 1uL gDNA/99uL H20
2. 30 pg/uL: 1.5 uL from (1)/ 48.5 uL H20
3. 3 pg/uL: 5 uL from (2)/ 45 uL H20
4. 300 fg/uL: 5 uL from (3)/ 45 uL H20
5. 30 fg/uL: 5 uL from (4)/ 45 uL H20
6. 3 fg/uL: 5 uL from (5)/ 45 uL H20
Concentrations 2-6 were used, and 1 blank tube of water was used for a total of 6
N6 primer dilution
Previously diluted by Sam
KOH preparation
1. Weigh a KOH pellet and put into a 1.5 mL tube
2. Add nuclease free H20 to get a 5M conc. (Note: MW KOH=56.11)
3. Cap and vortex
ALS Buffer Preparation
1. Add 82 uL H20, 10 uL 1M DTT, and 8 uL 5M KOH to 1.5 mL tube
2. Cap and vortex
NS Buffer Preparation
1. Add 3 mL H20, 3 mL 2M Tris buffer, 4 mL 1N HCl to 15 mL tube
2. Vortex
Master Mix Preparation
Use the following reagents for the master mix. Prepare in a .5 mL tube. The master mix was made for a total of 14 reactions.
1. 86.8 uL H20
2. 28 uL 10X repliphi phi-29 buffer
3. 70 uL 200 uM N6 primer
4. 11.2 uL 25 nM dNTP
5. 14 uL 2X SYBR green
6. 14 uL Repliphi phi-29
Add the enzyme last
To get volumes for a single reaction, divide by 14
Bio-Rad realtime PCR program
Line 1: 30 C, 6 min
Line 2: Plate read
Line 3: Go to line 1 for additional 99 times
Line 4: 85 C, 3 min
Line 5: 4 C, forever
Line 6: End
Procedures[edit]
1. After ALS and NS buffer is made, test pH (1:1 ratio). pH should be between 7-8.
2. UV NS and ALS buffer for 10 min
3. Put genomic DNA in two PCR strips with desired dilutions
4. Add 1.5 uL ALS to each tube and incubate at room temp for 3 min
5. Add 1.5 uL NS buffer to each tube and transfer to cool rack
6. Add 16 uL of master mix to each tube. Cap and vortex.
7. Put in Bio-Rad PCR machine and start program