Objectives
- PCR from extractions
- Primers (5 pM/μL) (new set because we are improving new protocol of standardization).
- Primer Forward= CGG GCC CCA TTA TTA TCA TGA CAT TAA CCT ATA AAA ATA GG
- It includes GGGCCC. Site for ApaI.
- Primer Reverse= CGA GCT CGC CTT TTG CTC ACA TCT TCT TTC C
- It includes GAGCTC. Site SacI.
PCR
- DNA was obtained from extractions and diluted(2μL DNA in 98μL H2O)
PCR amplification
Reactives
|
Pablo´s extraction
|
Tube 1
|
Tube 2
|
Buffer Standard Taq 10x NEB
|
5μL
|
5μL
|
5μL
|
Primer F
|
5μL
|
5μL
|
5μL
|
Primer R
|
5μL
|
5μL
|
5μL
|
10mM dNTPs
|
1μL
|
1μL
|
1μL
|
DNA
|
2μL
|
2μL
|
2μL
|
Taq
|
.5μL
|
.5μL
|
.5μL
|
H2O
|
31.5
|
31.5
|
31.5
|
Total
|
50μL
|
50μL
|
50μL
|
Control PCR
Reactives
|
-R
|
-F
|
-O
|
Buffer Standard Taq 10x NEB
|
2μL
|
2μL
|
2μL
|
Primer F
|
2μL
|
0μL
|
0μL
|
Primer R
|
0μL
|
2μL
|
0μL
|
.4mM dNTPs
|
10μL
|
10μL
|
10μL
|
DNA(Pablo's extraction)
|
2μL
|
2μL
|
2μL
|
Taq
|
.2μL
|
.2μL
|
.2μL
|
H2O
|
3.8
|
3.8
|
3.8
|
Total
|
20μL
|
20μL
|
20μL
|
Expectatives
- See in gel amplification of 3 extraction and not amplification in controls.
|