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1. Add 2.0 μl of digested mouse tail tip DNA to the appropriate PCR tube (see Protocol on Mouse Tail Tip Digest Protocol: Preparation of DNA).
2. Prepare enough of the following PCR Reaction Mixture for all the PCR reactions, based on 48 μl per PCR reaction. Add components sequentially.
39.8 μl of ddH2O
5 μl of 10X PCR Buffer with Mg2+
1 μl of 10 mM dNTP
1 μl 25 μM 5' End Primer
1 μl 25 μM 3' End Primer
0.2 μl of Taq Polymerase (added last)
3. Add 48 μl of PCR Reaction Mixture to each PCR tube.
4. Place tubes in a thermal cycler and run the program profile appropriate for the primers chosen.
5. Analyze PCR product on a 2% Agarose gel. (see Protocol on Analysis of DNA by Agarose Gel Electrophoresis)
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| 25 μM 3' End Primer |
| Synthesized specifically for gene of interest
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| 25 μM 5' End Primer |
| Synthesized specifically for gene of interest
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| 10mM dNTP |
| 10 mM dATP
10 mM dTTP
Store at -20°C.
10 mM dGTP
10 mM dCTP
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| PCR Buffer with Mg2+, 10X (use the buffer provided by Taq Polymerase manufacturer) |
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Oligonucleotide
dCTP
dTTP
DNA Polymerase, Taq
dGTP
dATP
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No hints are associated with this bioProtocol
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1. Modified from the Hanahan Laboratory at UCSF.
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