05/22/2015
SYBR qPCR to determine plasmid counts for dilution experiment
| pX330 primer |
P209/210 (G)
|
| internal primer |
18S (C)
|
| Dilution |
2ng/rxn
|
Step 1: make two PCR tubes for each sample with the following volumes of water and gDNA for 2ng of gDNA per well
| '
|
1rxn
|
x3.5 = 7ng
|
| gDNA |
x |
|
| water |
to 4.5 |
|
| Total |
4.5 |
15.75
|
| Tube label
|
Cell type
|
gRNA
|
ug DNA
|
ul lipo
|
rep
|
ng/µL
|
dilute 1:20
|
ul dilution
|
ul water
|
| 1 |
Gal4-EED |
g034 |
0.5 |
3 |
1 |
81.058 |
4.0529 |
1.73 |
14.02
|
| 2 |
Gal4-EED |
g034 |
0.5 |
3 |
2 |
86.697 |
4.33485 |
1.61 |
14.14
|
| 3 |
Gal4-EED |
g034 |
0.5 |
5 |
1 |
23.89 |
1.1945 |
5.86 |
9.89
|
| 4 |
Gal4-EED |
g034 |
0.5 |
5 |
2 |
24.767 |
1.23835 |
5.65 |
10.10
|
| 5 |
Gal4-EED |
g034 |
1 |
3 |
1 |
85.32 |
4.266 |
1.64 |
14.11
|
| 6 |
Gal4-EED |
g034 |
1 |
3 |
2 |
70.244 |
3.5122 |
1.99 |
13.76
|
| 7 |
Gal4-EED |
g034 |
1 |
5 |
1 |
13.387 |
0.66935 |
10.46 |
5.29
|
| 8 |
Gal4-EED |
g034 |
1 |
5 |
2 |
23.213 |
1.16065 |
6.03 |
9.72
|
| 9 |
water |
|
|
|
|
|
|
|
15.75
|
Step 2: make primer master mixes. Will have to add 3.5x to each triplicate gDNA tube so can calculate mastermixes from that
- 8 samples, 2 tubes/sample, 1 tube for no template control, total of 17 tubes
| '
|
1rxn
|
3.5/triplicate tube
|
17.7
|
| primer |
3 |
10.5 |
185.85
|
| SYBR |
7.5 |
26.25 |
464.625
|
| Total |
10.5 |
36.75 |
650.475
|
Step 3: mix gDNA and primer mixes
- Add 36.75ul primer mix to each sample tube
Step 4: pipette plate
- mix tubes immediately before pipetting, add 15 ul from each tube into 3 wells
| '
|
1-3
|
4-6
|
7-9
|
10-12
|
| A |
__1 G__ |
__1 C__ |
blank-G |
______
|
| B |
__2 G__ |
__2 C__ |
blank-C |
______
|
| C |
__3 G__ |
__3 C__ |
______ |
______
|
| D |
__4 G__ |
__4 C__ |
______ |
______
|
| E |
__5 G__ |
__5 C__ |
______ |
______
|
| F |
__6 G__ |
__6 C__ |
______ |
______
|
| G |
__7 G__ |
__7 C__ |
______ |
______
|
| H |
__8 G__ |
__8 C__ |
______ |
______
|
| Cell type
|
Plasmid 1
|
Plasmid 2
|
Rep
|
260
|
280
|
260/280
|
ng/µL
|
1:20 dil
|
ul dil for 7ng
|
ul water
|
| Luc14 |
KAH226 |
g034 |
1 |
0.081 |
0.04 |
2.021 |
80.531 |
4.02655 |
1.738460965 |
14.01153903
|
| Luc14 |
KAH226 |
g034 |
2 |
0.126 |
0.061 |
2.06 |
126.451 |
6.32255 |
1.107148223 |
14.64285178
|
| Luc14 |
KAH226 |
g034 |
3 |
0.091 |
0.044 |
2.045 |
90.862 |
4.5431 |
1.540798133 |
14.20920187
|
| Luc14 |
GFP |
g034 |
1 |
0.104 |
0.051 |
2.052 |
104.239 |
5.21195 |
1.343067374 |
14.40693263
|
| Luc14 |
GFP |
g034 |
2 |
0.065 |
0.031 |
2.094 |
64.965 |
3.24825 |
2.155006542 |
13.59499346
|
| Luc14 |
GFP |
g034 |
3 |
0.039 |
0.02 |
1.99 |
39.096 |
1.9548 |
3.580928995 |
12.169071
|
|
|
|
|
|
|
|
|
|
|
15.75
|
|
Project name
