Amplification of bisulfite converted input DNA library[edit]
Use half of the library to perform amplification
PCR Amplification of input DNA library[edit]
|
1 rxn |
Prepare 4 rxns
|
DNA |
5 |
|
H2O |
36.6 |
161.04
|
PCR_F (10uM) |
4 |
17.6
|
PCR_R (10uM) |
4 |
17.6
|
SYBR 50X |
0.4 |
1.76
|
2X Fusion Enzyme |
50 |
220
|
Total |
100 |
440
|
|
- Add 95ulmix to 5ul library
Setup
|
5ng
|
10ng
|
50ng
|
100ng
|
|
perform
- 98C 30sec
- 98C 10 sec
- 64C 20 sec
- 72C 20sec
- plate read
- line 2 4 more times
- 98C 10sec
- 72C 20sec
- plate read
- goto line 7 5 more times
- 72C 5 min
- 15C hold
Gel Quantification[edit]
- with input DNA and low input, bisulfite converted, PCR amplified DNA library
File:ZhangLab 2 2010-03-07 15hr 50min.jpg
Conclusion[edit]
- Due to bad experimental design with no positive and negative control, I obtained none useful information through this experiment.
- No +ve control,
- No -ve control
- It could be the adapter annealing problem (solved by +ve control + qpcr after ligation)
- It could be the self annealing of adapter by the adapter
- The primer used for amplification of methylation adapter might not be the right one.(update primer need to be used is the PCR primer PR 1.0 and PCR primer PE2.0 which is the one I used PCR_F and PCR_R)
Improvements[edit]
- For all experiments from now on include a +&-control.
- Include +ve and -ve control for the experiment
- Quantify after adapter ligation
- Use 5ul of 1/10x adapter to do annealing