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PCR: Lochy (amplifluor)
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Export PDF
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| ID:
291
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Amplifluor:
Yes
If you require further assistance regarding this PCR protocol, please contact us at genotyping.support@apf.edu.au
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| Nucleotide sequence (5´ ⇒ 3´) |
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| Primer name |
Sequence |
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Lochy-1
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GAAGGTGACCAAGTTCATGCTTTGGGTTCCTTGAAGCCCTGATCT
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Lochy-2
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GAAGGTCGGAGTCAACGGATTTGGGTTCCTTGAAGCCCTGATCAA
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Lochy-R
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ATGACTGTTCTCTTTGTGTGTT
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| Reagents / Constituents |
Commercial name |
Stock
Concentration |
20x Primer mix
concentration (μM) |
Volume (μL) |
| DNA Sample |
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NA ng/μl
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2.0
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| 10x Buffer |
10x Reaction Mix S Plus
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NA mM MgCl2
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1.0
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| dNTPs |
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2.5 mM
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0.8
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| 20x amplifluor SNP FAM Primer |
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NA μM
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NA
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| 20x amplifluor SNP JOE Primer |
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NA μM
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NA
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| Primer:
Lochy-1
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NA μM
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0.5
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| Primer:
Lochy-2
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NA μM
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0.5
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| Primer:
Lochy-R
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NA μM
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7.5
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| Amplifluor Primer Volume |
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0.5
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| Water |
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4.6
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| Taq Polyermase |
Platinum Taq (5U/µl)
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0.1
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| Comments on protocol: |
DNA Sample (cleaned ear punch in dH20-see cleanup protocol).
The reaction needs to be done at room temperature with reagents added in order (dh2O, dNTPs, SNP FAM, SNP JOE, Lochy, 10x reaction mix, Taq, DNA sample).
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| Steps |
Temp (°C ) |
Time (seconds) |
| Hot start:
true
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| Initiation/Melting |
94.0
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240
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| Denaturation |
94.0
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10
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| Annealing |
55.0
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20
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| Elongation |
72.0
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40
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Number of cycles
(repeat denature, anneal & elongate):
35
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| Strand completion (i.e. 72°C, 10 min) |
72.0
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180
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| Finish (i.e. 4°C, indefinite) |
10.0
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Hold
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| Picture |
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