(r2) Barrick Lab :: ProceduresTargetedSequencing

---+++ Primer Design

Primers sequences and sometimes stocks already exist for some genes: _[[ProceduresSequencingTopA][topA]]_

---+++ PCR reaction

---+++ 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 or Qiagen kit or similar reagent. Be sure to elute in buffer compatible with DNA sequencing applications. (Buffer 6 for the GE kit, or ddH<sub>2</sub>O)

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.

Barrick Lab > ProtocolList > ProceduresTargetedSequencing

Contributors to this topic

JeffreyBarrick, CameronRoots, DanielDeatherage, GabrielSuarez

Topic revision: r2 - 2009-08-05 - 14:30:19 - Main.JeffreyBarrick