PCR – C.pugetti gDNA from 20090526

This is a repeat of yesterday’s PCR due to the presence of bands in the water-only samples. Will use reagents and universal 16s bacterial primers (27F & 1492R) provide by the Horner-Devine lab in hopes of: 1) getting this two work and, 2) figuring out the source of the contamination.

All rxns were prepared sterily and all instruments, racks, tubes, tips and water were UV-sterilized for ~45mins in the biological hood. Rxns were prepared in the biological hood. PCR setups are here. Anneal 60C. Cycling params same as yesterday.

Lane 1 – 100bp ladder

Lane 2 – DNA (HD Rxn 1)

Lane 2 – H2O (HD Rxn 1)

Lane 3 – H2O (HD Rxn 1)

Lane 4 – DNA (HD Rxn 2)

Lane 5 – H2O (HD Rxn 2)

Lane 6 – H2O (HD Rxn 2)

Lane 7 – DNA (SR Rxn)

Lane 8 – H2O (SR Rxn)

Lane 9 – H2O (SR Rxn)

Lane 10 – 100bp ladder

Results: Well, we got our band and NO contamination in any H2O lanes. The super-bright, 1500bp band will be excised and purified using Millipore spin columns and submitted for sequencing. However, this gel is interesting because the primers provided by Mike (used in HD Rxn 1 and SR Rxn) did not amplify anything…

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