| |
|
|
PCR: KENOBI (amplifluor)
|
Export PDF
|
| ID:
257
|
|
Amplifluor:
Yes
If you require further assistance regarding this PCR protocol, please contact us at genotyping.support@apf.edu.au
|
|
|
| Nucleotide sequence (5´ ⇒ 3´) |
|
| Primer name |
Sequence |
|
Kenobi-1
|
GAAGGTGACCAAGTTCATGCTTGCAGGAACCCTAATATCACATGGTGA
|
|
Kenobi-2
|
GAAGGTCGGAGTCAACGGATTGCAGGAACCCTAATATCACATGGTGG
|
|
Kenobi-R
|
TGATGTTAGACTGAAGGCTGAA
|
|
|
|
|
| Reagents / Constituents |
Commercial name |
Stock
Concentration |
20x Primer mix
concentration (μM) |
Volume (μL) |
| DNA Sample |
|
NA ng/μl
|
|
2.0
|
| 10x Buffer |
10x Reaction Mix S Plus
|
NA mM MgCl2
|
|
1.0
|
| dNTPs |
|
2.5 mM
|
|
0.8
|
| 20x amplifluor SNP FAM Primer |
|
NA μM
|
|
NA
|
| 20x amplifluor SNP JOE Primer |
|
NA μM
|
|
NA
|
| Primer:
Kenobi-1
|
|
NA μM
|
1.5
|
|
| Primer:
Kenobi-2
|
|
NA μM
|
0.5
|
|
| Primer:
Kenobi-R
|
|
NA μM
|
22.5
|
|
| Amplifluor Primer Volume |
|
|
|
0.5
|
|
| Water |
|
|
|
3.9
|
| Taq Polyermase |
Platinum Taq (5U/µl)
|
|
|
0.1
|
|
| Comments on protocol: |
DNA Sample (cleaned ear punch in dH20-see cleanup protocol).
Reactions to be done at room temperature with reagents added in order.
Final MgCl2 conc. is 2.5mM.
Reagent order: dH2O, DNTPs, 20x amplifluor SNP FAM Primer, 20x amplifluor SNP JOE Primer, 20x Kenobi specific primer mix, 10x Reaction Mix S Plus, 10mM MgCl, Platinum Taq (5u/ul), DNA sample.
|
|
|
| Steps |
Temp (°C ) |
Time (seconds) |
| Hot start:
true
|
|
|
| Initiation/Melting |
94.0
|
240
|
| Denaturation |
94.0
|
10
|
| Annealing |
60.0
|
20
|
| Elongation |
72.0
|
40
|
Number of cycles
(repeat denature, anneal & elongate):
30
|
|
|
| Strand completion (i.e. 72°C, 10 min) |
72.0
|
180
|
| Finish (i.e. 4°C, indefinite) |
10.0
|
Hold
|
|
|
|
| Picture |
|
Database
