Difference between revisions of "Standard PCR"
From GcatWiki
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| − | A. Reaction Components: | + | A. Reaction Components: |
2 ul 10X PCR buffer w/ 15 mM MgCl2 | 2 ul 10X PCR buffer w/ 15 mM MgCl2 | ||
| Line 19: | Line 19: | ||
Creat masta mix if desired | Creat masta mix if desired | ||
| − | B. Thermal Profile | + | B. Thermal Profile |
1 circle of : 94 C 4 mins | 1 circle of : 94 C 4 mins | ||
| − | 30 circles of: | + | 30 circles of: 94 C 15 sec.; annealing temp. 15 sec.; 74 C 30 sec. |
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1 circle of : 74 C 4 mins | 1 circle of : 74 C 4 mins | ||
annealing temp. should be about 4 C below the lower of the Tm values for the two primers. | annealing temp. should be about 4 C below the lower of the Tm values for the two primers. | ||
Revision as of 19:06, 9 June 2008
A. Reaction Components:
2 ul 10X PCR buffer w/ 15 mM MgCl2
2 ul 2 mM dNTPs (working concentration 2 mM each)
1 ul forward primer (20 pmol)
1 ul reverse primer (20 pmol)
1 ul Taq DNA polymerase
12 ul dH2O
19 ul total
add 1 ul template DNA
Creat masta mix if desired
B. Thermal Profile
1 circle of : 94 C 4 mins
30 circles of: 94 C 15 sec.; annealing temp. 15 sec.; 74 C 30 sec.
1 circle of : 74 C 4 mins
annealing temp. should be about 4 C below the lower of the Tm values for the two primers.
