Home | Achievement | Programmes | Projects | Experts | Staffs | Publications | Journals |
Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
MOLECULAR BIOLOGY: WORKING WITH DNA
POLYMERASE CHAIN REACTION
MOUSE TAIL TIP DIGEST PROTOCOL: PCR FROM TAIL TIP DNA
Mouse Tail Tip Digest Protocol: PCR from Tail Tip DNA
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)
25 μM 3' End Primer Synthesized specifically for gene of interest 25 μM 5' End Primer Synthesized specifically for gene of interest 10mM dNTP 10 mM dATP
10 mM dTTP
Store at -20°C.
10 mM dGTP
10 mM dCTP
PCR Buffer with Mg2+, 10X (use the buffer provided by Taq Polymerase manufacturer) BioReagents and Chemicals
DNA Polymerase, Taq
No hints are associated with this bioProtocol Citation and/or Web Resources
1. Modified from the Hanahan Laboratory at UCSF.