---+ Targeted PCR and Sanger Resequencing of Mutations ---++ Primer Design Use the NCBI primer-blast tool to design primers: http://www.ncbi.nlm.nih.gov/tools/primer-blast/ In the "PCR Template" box, enter "NC_012967" as the accession to search. This is the REL606 genome. Select appropriate coordinates for each primer. Default settings for melting temperatures, etc, are fine. Under "organism" in the "Primer Pair Specificity Checking Parameters" section, enter "Escherichia coli (taxid:562)". *For point mutations and small indels:* Design two primers ~200-350 bp upstream and ~200-350 bp downstream of each mutation to amplify a 400-700 bp fragment. Sanger sequence from only one end. One reaction per template. *For IS-insertions:* Design primers ~200-350 bp upstream and downstream of ends of mutation to amplify product. Do control reactions with REL606 to verify the expected size change. Sequence from both sides for IS-insertions so that we can determine the new IS orientation and new junction sequences. *For large deletions (>4kb):* Design primers ~200-350 bp upstream and downstream of ends of mutation to amplify a product for the deletion. If the product for the non-deleted ancestral genome is too large to reliably amplify (> 5 kb), then order an additional primer within the deleted interval that can amplify a 400-700 bp fragment with the original forward primer. Always do control reactions with REL606 to verify that a band appears in it for just one primer pair (the non-deleted specific pair) and that a band appears for the evolved genomes with just one pair (the deletion specific pair). Sequence the deletion from just one end. Be sure to design primer pairs such that the products of each PCR differ by >200 bp in length, so that you can tell them apart on the gel! *For rearrangements, amplifications, and inversions* Design primers ~200-350 bp upstream and downstream of new junctions. PCR across the junction and Sanger sequence to verify the exact junction site. Be careful to NOT design primers in repetitive regions (such as IS elements, tRNA genes, rRNA genes, etc.) that could give confusing amplification products! Primers sequences and stocks already exist for some genes: _[[ProceduresSequencingTopA][topA]]_ ---++ PCR reaction Perform [[ProceduresStandardPCR][standard PCR]] reactions. In all PCR reactions use ~1 ng/ul of genomic DNA sample as template. Always use high-fidelity polymerase. Possibly "long-template" polymerase if the amplified fragments are very large (>5 kb). ---++ Check PCR and Quantify DNA Yields Run 2-5 µl of sample + 2-5 µl of glycerol load buffer on a 0.8-2% agarose gel. Load 5 µl of 0.1µg/ml DNA ladder. Use either 100-bp or 1-kb DNA ladder size as appropriate. Ladders commonly used in lab. | *Link* | *Company* | *Description* | *Cat #* | *Unit* | *Price* | | [[http://www.neb.com/nebecomm/products/productN3231.asp][%ICON{external}%]] | New England Biolabs | 100-bp ladder | <nop>N3231L | 500 gel lanes | $212 | Estimate band concentrations by eye or by saving the image in TIFF format and finding band densities with [[http://rsbweb.nih.gov/ij/][ImageJ]]. Compare to a reference band of similar density and determine the concentration of the original sample. ---++ DNA purification Use illustra™ GFX™ PCR DNA and Gel Band purification kit, Qiagen kit, or similar kit. Be sure to elute in buffer compatible with DNA sequencing applications. (Buffer 6 for the GE kit, or ddH<sub>2</sub>O) Do NOT elute in 1×TE. For all practical purposes assume 90% recovery of the input DNA sample as long as it is >200 bp in length. Smaller fragments are not bound as efficiently by the column. ---++ Sequencing Reaction Normal submissions to RTSF at MSU should be in 96-well plates.
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Topic revision: r5 - 2010-09-27 - JeffreyBarrick