|
|
PCR: Twimp_(Twp)
|
Export PDF
|
ID:
356
|
|
Amplifluor:
No
|
|
Nucleotide sequence (5´ ⇒ 3´) |
|
Primer name |
Sequence |
TwpF'
|
GAAAGAAACCACCATTTTCTGAT
|
TwpR'WT
|
TTCTGGACTTGGTACAGTATGACAGGCA
|
TwpR'M
|
GGGCAGAGATCTGCAGCAGAAGCCTGGACTTGGTACAGTATGACATACC
|
|
|
|
Reagents / Constituents |
Commercial name |
Stock Concentration |
20x Primer mix concentration (μM) |
Volume (μL) |
DNA Sample |
|
NA ng/μl
|
|
1.0
|
10x Buffer |
10x 0289 PCR Buffer
|
0.0 mM MgCl2
|
|
5.0
|
dNTPs |
|
10.0 mM
|
|
0.5
|
20x amplifluor SNP FAM Primer |
|
NA μM
|
|
NA
|
20x amplifluor SNP JOE Primer |
|
NA μM
|
|
NA
|
Primer:
TwpF'
|
|
10.0 μM
|
NA
|
3.0
|
Primer:
TwpR'WT
|
|
10.0 μM
|
NA
|
3.0
|
Primer:
TwpR'M
|
|
10.0 μM
|
NA
|
3.0
|
Amplifluor Primer Volume |
|
|
|
NA
|
|
Water |
|
|
|
27.35
|
Taq Polyermase |
|
|
|
0.15
|
100mM MgCl2
|
|
|
|
1.75
|
|
Comments on protocol: |
DNA Sample (ear punch).
Final conc. of 3.5mM MgCl2
|
|
|
Steps |
Temp (°C ) |
Time (seconds) |
Hot start:
false
|
|
|
Initiation/Melting |
94.0
|
120
|
Denaturation |
94.0
|
20
|
Annealing |
65.0
|
20
|
Elongation |
72.0
|
20
|
Number of cycles (repeat denature, anneal & elongate):
35
|
|
|
Strand completion (i.e. 72°C, 10 min) |
72.0
|
180
|
Finish (i.e. 4°C, indefinite) |
10.0
|
Hold
|
|
|
|
% Agarose |
3.0
|
Volt |
400.0
|
Estimated Running Time (min) |
30.0
|
Number |
Band (bp) |
Genotype |
1. |
195
|
Wt/Wt
|
2. |
215
|
Twp/Twp
|
|