G.tigrina Hox gene DthoxC insertion into prokaryote E.coli / (by UNIamCloning)/2011/09/22

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DNA Extraction and Purification of G.tigrina (cont.) 9/22/2011

  • The DNA extraction of the sample (Gt.1) was checked for success using an electrophoresis gel and a Qubit Fluorometric Quantifier. A 5 µl RE digest sample containing 5 µl DNA extract, 2.5 µl H2O, 0.5 µl FastDigest PSTI enzyme, and 2.0 µl 10x conc. FastDigest buffer, was vortexed and incubated at 37°C for 30 min. and run on the 1.8% agarose gel in TBE buffer at 150 V for 50 min. In adjacent wells on the gel the RE digest sample was a negative control of 5 µl DNA extract (undigested) and 100+bp ladder.

The gel more or less failed; the ladder showed up fine in its lane, but the RE digest and negative control produced only a faint smear and no bands. The Qubit was then used to quantify exactly what the concentration of DNA was in the (Gt.1) sample, and the result showed 2.80 µg/mL. (This low concentration is probably why the samples did not appear on the gel.)

Finally, we decided to experiment with the amount of planarian tissue used during the extraction process. We started new extractions on starved and halved planarians, but this time we tried half the amount of tissue as in (Gt.1), as well as twice the amount of tissue. To do this we put one half of one planarian in one tube labeled (Gt.2-1), the other half of the same planarian in a tube labeled (Gt.2-2), and all four halves of two whole planarians into a third tube labeled (Gt.3). All three tubes were placed into an incubator set at 56°C and allowed to incubate overnight.