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Last updated |
| August 10, 2003 | |
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Polymerase Chain Reaction (with Pfu)
Polymerase Chain Reaction (PCR) was used for amplification of 16S rRNA gene from the community DNA from the environments and pure cultures. Proofreading polymerase Pfu is used in order to obtain high fidelity product and also to facilitates the cloning into the TOPO cloning vector directly.
Example of primers used for PCR are listed below:
Primers used for amplification and sequencing
of the 16S rRNA gene
27F 5' AGAGTTTGATCCTGGCTCAG 3'
533F 5' GTGCCAGCMGCCGCGGTAA 3'
981R 5´GGG TTG CGC TCG TTG CGG G 3´
1492R 5´GGTTACC TTG TTA CGA CTT3´
T3 5' AATTAACCCTCACTAAAGGGA 3'
T7 5' TAATACGACTCACTATAGGG 3'
Setting up PCR Reaction:
| 10x Pfu Buffer | 5.0 ml |
| DMSO | 3.0 ml |
| Primer 1 | 1.5 ml |
| Primer 2 | 1.5 ml |
| dNTP | 3.0 ml |
| DNA | 1.0-5.0 ml |
| dH20 | add to 49.0 ml |
| HOT START: | |
| Pfu Polymerase | 1.0 ml |
| Total | 50 ml |
Set up thermocyler program:
Hot Start
Denaturation 94°C 30 sec
Annealing 55°C 30 sec
Extension 72°C 90 sec (1 min/kb)
25-30 cycles
After cycling, incubate the reaction at 72°C for another 7 min. Cool
the reactions to 4°C, ready for further experiments.