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PCR: BLOBBY
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Export PDF
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| ID:
175
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Amplifluor:
No
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| Nucleotide sequence (5´ ⇒ 3´) |
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| Primer name |
Sequence |
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Blobby F
|
AAAGCCCCACATGTAGATCG
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Blobby R
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TGAGGTATATGCTGAACCTCTTAT
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| Reagents / Constituents |
Commercial name |
Stock
Concentration |
20x Primer mix
concentration (μM) |
Volume (μL) |
| DNA Sample |
|
NA ng/μl
|
|
0.1
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| 10x Buffer |
|
20.0 mM MgCl2
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|
5.0
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| dNTPs |
|
10.0 mM
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|
1.0
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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 |
|
NA μM
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NA
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| Primer:
Blobby F
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|
10.0 μM
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NA
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1.0
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| Primer:
Blobby R
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|
10.0 μM
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NA
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1.0
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| Amplifluor Primer Volume |
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NA
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| Water |
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40.45
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| Taq Polyermase |
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0.05
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Triton
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0.5
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| Comments on protocol: |
DNA Sample (from ear punch).
Mutagenic primer introduces Psi1 restriction site in the wt but not the mutant Alms1 sequence.
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| Steps |
Temp (°C ) |
Time (seconds) |
| Hot start:
false
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| Initiation/Melting |
94.0
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120
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| Denaturation |
94.0
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30
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| Annealing |
59.0
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60
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| Elongation |
72.0
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120
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Number of cycles
(repeat denature, anneal & elongate):
38
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| Strand completion (i.e. 72°C, 10 min) |
72.0
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300
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| Finish (i.e. 4°C, indefinite) |
10.0
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Hold
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| Enzyme name: |
PsiI
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| Concentration: |
NA units/μl
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| Company: |
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| Pcr volume: |
20.0 μl
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| Enzyme volume: |
1.0 μl
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| Buffer volume: |
3.0 μl
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| Water volume: |
3.0 μl
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| Temperature: |
37.0 °C
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| Time: |
120.0 mins
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| Comments: |
Additional reagent: 10x BSA, 3μl.
10x Buffer (SEB Buffer B).
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| % Agarose |
4.0
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| Volt |
300.0
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| Estimated Running Time (min) |
30.0
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| Number |
Band (bp) |
Genotype |
| 1. |
200
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all uncut
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| 2. |
190
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PsiI cut wt
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| 3. |
200
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PsiI cut Mutant
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